Protein-polymer nanoassemblies and intracellular protein delivery

ABSTRACT

The invention provides novel polymer-protein conjugates and molecular assemblies for controlled intracellular delivery of proteins, and compositions and methods of preparation and use thereof.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/876,441, filed Jul. 20, 2019, the entire content ofeach of which is incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberGM128181. awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to polymer-protein conjugates andprotein delivery. More particularly, the invention relates to molecularassemblies of polymer-protein conjugates and controllable intracellulardelivery of proteins in response to specific microenvironment, andcompositions and methods of preparation and use thereof.

BACKGROUND OF THE INVENTION

Reaction between large molecules is an inherently slow process,especially when one of the reactive components involve functional groupson protein surfaces, because the reactive functionalities areheterogeneously distributed in low densities across large surfaces.Compared to the classical small molecule-based organic reactions thatare typically carried out at high mM concentrations, proteins have lowerreaction concentration limits (μM) and lower conjugation efficiency. Inaddition, the larger sizes of the reactive components would contributeto slow diffusion rates and low collision frequency. (White, et al. ACSCent. Sci. 2018, 4, 197-206.)

Programmable polymer-protein conjugates have implications in severalapplications, including sensing in complex environments, enzymecatalysis in incompatible media, and delivery of biologics. (Cobo, etal. Nat. Mater. 2015, 14, 143-159; Droumaguet, et al. Angew. Chem. Int.Ed. 2008, 47, 6263-6266; Huang, et al. Nat. Commun. 2013, 4, 2239;Velonia, et al. J. Am. Chem. Soc. 2002, 124, 4224-4225; Nguyen, et al.Nat. Chem. 2013, 5, 221-227; Baslé, et al. Chem. Biol. 2010, 17,213-227; Brodin, et al. J. Am. Chem. Soc. 2015, 137, 14838-14841;Danial, et al. J. Am. Chem. Soc. 2014, 136, 8018-8026; Dutta, et al. J.Am. Chem. Soc. 2017, 139, 5676-5679; Ventura, et al. Biomacromolecules2015, 16, 3161-3171.)

Despite its potential in several diseases, transporting proteins acrossthe cellular membrane remains a challenge that requires urgentattention. As proteins orchestrate most of the critical cellularprocesses, imbalance in the activity of intracellular proteins forms thebasis for many human diseases. Although straightforward in principle tosimply use the deficient protein itself as the therapeutic, such anapproach is complicated by the fact that proteins are structurallyfragile in non-native environments and are impermeable to cellularmembrane. Several protein delivery systems are being developed tomitigate these risks. However, vast majority of these approaches havefocused on extracellular targets, such as with delivering antibodies tocell surfaces and addressing insulin deficiencies. (D'Astolfo, et al.Cell 2015, 161, 674-690; Zhou, Het al. Cell Stem Cell 2009, 4, 381-384;Jakka, et al. Angew. Chem. Int. Ed. 2019, 58, 7713-7717; Yin, et al.Nat. Biotechnol. 2016, 34, 328-333; Zuris, et al. Nat. Biotechnol. 2014,33, 73-80; Jo, et al. Nat. Med. 2005, 11, 892-898; Cardinale, et al.Trends Mol. Med. 2008, 14, 373-380; Mo, et al. Chem. Soc. Rev. 2014, 43,3595; Leader, et al. Nat. Rev. Drug Discov. 2008, 7, 21-39; Pavlou, etal. Nat. Biotech. 2004, 22, 1513-1519; Caravella, et al. Curr. OpinionChem. Biol. 2010, 14, 520-528.)

There have been previous reports of encapsulating and deliveringproteins. Encapsulation using polyelectrolyte complexes and liposomalassemblies constitute two of the major approaches. The polyelectrolytemethod is simple and fast, which typically utilizes a positively chargedpolymer or nanoparticle to bind to a negatively charged protein. (Zuris,et al. Nat. Biotechnol. 2014, 33, 73-80; Salmaso, et al. Int. J. Pharm.2013, 440, 111-123; Scaletti, et al. Chem. Soc. Rev. 2018, 47,3421-3432; Liu, et al. ACS Appl. Mater. Interfaces 2017, 9, 2023-2028;Fegan, et al. Chem. Rev. 2010, 110, 3315-3336; Luo, et al. Chem. Rev.2016, 116, 13571-13632; Dun, et al. J. Am. Chem. Soc. 2017, 139,13960-13968; Matsuurua, et al. RSC Adv. 2014, 4, 2942-2953; Doolan, etal. Chem. Eur. J. 2018, 24, 984-991; Gu, et al. Chem. Soc. Rev. 2011,40, 3638-3655; Lu, et al. J. Controlled Release 2014, 194, 1-19; Lam, etal. Biomacromolecules 2016, 17, 2820-2829; Lee, et al. Angew. Chem.,Int. Ed. 2009, 48, 5309-5312; Ghosh, et al. J. Am. Chem. Soc. 2010, 132,2642-2645; González-Toro, et al. J. Am. Chem. Soc. 2012, 134,6964-6967.)

As the overall surface charge of these complexes is positive, theyexhibit the tendency to be transported across the negatively chargedcellular membrane. However, these complexes with positive surfacecharges do tend to suffer from non-specific fouling by serum proteinsand associated toxicities. (Lv, et al. J. Controlled Release 2006, 114,100-109; Fröhlich, et al. Int. J. Nanomedicine 2012, 7, 5577-5591;Intra, et al. J. Controlled Release 2008, 130, 129-138.)

Liposome surfaces, on the other hand, can be made to avoid non-specificfouling by using charge-neutral lipids, but the amount of proteins thatcan be loaded in a unit volume of these assemblies tends to be quitelimited. This is due to the lack of a driving force for thewater-soluble proteins to be sequestered within the aqueous lumen of theliposomes, compared to the bulk aqueous phase. (Swaminathan, et al.Expert Opin. Drug Delivery 2012, 9, 1489-1503; Chatin, et al. Mol. Ther.Nucleic Acids 2015, 4, e244.)

Covalent conjugation of polymeric molecules to proteins has beenexplored, especially in the context of stabilizing the latter, a popularexample being the so-called PEGylation of proteins. Earlier approachesto attaching polymers to proteins involved the formation of stableconjugates, where the success metrics relied on whether the modificationaffected the native activity of the protein. (Cobo, et al. Nat. Mater.2015, 14, 143-159; Droumaguet, et al. Angew. Chem. Int. Ed. 2008, 47,6263-6266; Huang, et al. Nat. Commun. 2013, 4, 2239; Velonia, et al. J.Am. Chem. Soc. 2002, 124, 4224-4225; Nguyen, et al. Nat. Chem. 2013, 5,221-227; Baslé, et al. Chem. Biol. 2010, 17, 213-227; Brodin, et al. J.Am. Chem. Soc. 2015, 137, 14838-14841; Danial, et al. J. Am. Chem. Soc.2014, 136, 8018-8026; Dutta, et al. J. Am. Chem. Soc. 2017, 139,5676-5679; Ventura, et al. Biomacromolecules 2015, 16, 3161-3171; Gu, etal. Chem. Soc. Rev. 2011, 40, 3638-3655; Mummidivarapu, et al.Bioconjugate Chem. 2018, 29, 3999-4003; Khondee, Set al.Biomacromolecules 2011, 12, 3880-3894; Ellis, et al. J. Am. Chem. Soc.2012, 134, 3631-3634; Rudolph, et al. In Protein Function: A PracticalApproach, 2nd ed.; Creighton, T. E., Ed.; Oxford: New York, 1997; p 64.)

As these modifications did affect protein activities in many cases,there have been a few reports that introduced conjugation throughreversible chemical bonds. Despite the promise and many great advancesin efficient organic synthetic methodologies, there is a dearth ofsuccessful methodologies for the transmembrane transport of activeproteins using these approaches. (Dutta, et al. J. Am. Chem. Soc. 2017,139, 5676-5679; Ventura, et al. Biomacromolecules 2015, 16, 3161-3171;Chiper, et al. Adv. Healthcare Mater. 2018, 7, 1701040; Qian, et al.Angew. Chem. Int. Ed. 2018, 57,1532-1536; Wang, et al. Angew. Chem.,Int. Ed. 2014, 53, 13444-13448.)

Accordingly, an ongoing need remains for an effective delivery vehiclefor proteins, one that is preferably capable of traceless release ofproteins that retain their native activities permitting practicaltherapeutic applications.

SUMMARY OF THE INVENTION

The invention provides a novel delivery platform for intracellulardelivery of proteins that offers rapid and reversible conjugationcapabilities. Surface modified proteins can be rapidly conjugated withpolymers, which can be fully reversed in the presence of a specific andbiologically relevant stimulus at the delivery site, e.g., reactiveoxygen species, reducing environment, or variations in pH. The utilityof this self-assembly process is demonstrated with intracellulardelivery of proteins with retained function.

Modifications in the linker chemistry offers the ability to triggerthese assemblies with various chemical inputs. Efficient formation ofnanoassemblies based on polymer-protein conjugates has implications in avariety of areas at the interface of chemistry with materials andbiology, such as in the generation of active surfaces and in delivery ofbiologics.

In one aspect, the invention generally relates to a functionalizedcopolymer, comprising: a first monomer of PEG-methacrylate (PEG-MA); anda second monomer of methacrylate having a side chain modified with asalicylhydroxamate moiety.

In another aspect, the invention generally relates to a surface modifiedprotein comprising arylboronic acid modifications of one or more lysineresidues.

In yet another aspect, the invention generally relates to apolymer-protein conjugate, comprising: a copolymer comprising a firstmonomer of PEG-methacrylate (PEG-MA) and a second monomer ofmethacrylate having a side chain conjugated to a protein via adegradable linker.

In yet another aspect, the invention generally relates to a molecularassembly comprising the polymer-protein conjugate disclosed herein.

In yet another aspect, the invention generally relates to a compositioncomprising the molecular assembly disclosed herein.

In yet another aspect, the invention generally relates to a method fordelivering a protein, comprising: surface functionalizing the proteinwith arylboronic acid modifications of one or more lysine residues onthe protein; forming a polymer-protein conjugate by reacting the surfacefunctionalized protein with a copolymer comprising a first monomer ofPEG-methacrylate (PEG-MA) and a second monomer of methacrylate having aside chain modified by a salicylhydroxamate moiety thereby forming amolecular assembly comprising the polymer-protein conjugate, wherein thepolymer-protein conjugate comprises a degradable linker; transportingthe molecular assembly to a target site to degrade the linker (and thusthe molecular assembly) thereby releasing the protein at the targetsite.

In yet another aspect, the invention generally relates to a method forforming a molecular assembly, comprising: surface functionalizing theprotein with arylboronic acid modifications of one or more lysineresidues on the protein; and reacting the surface functionalized proteinwith a copolymer comprising a first monomer of PEG-methacrylate (PEG-MA)and a second monomer of methacrylate having a side chain modified by asalicylhydroxamate moiety thereby forming a molecular assemblycomprising the polymer-protein conjugate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Polymer-protein assembly formation and disassembly process.Modification of lysine surface functional groups in proteins witharylboronic acid; Protein-polymer complexation through a reversibleclick reaction between protein and polymer; Stimuli induced disassemblyof the protein-polymer complex to release the protein with a tracelessform.

FIG. 2 . Protein-polymer complex formation. a) SDS-PAGE gel forcomplexation between modified RNaseA and polymer P1 at 29 μMconcentration of RNaseA; b) Complexation kinetics monitored throughtemporal evolution of the free protein concentration upon mixingmodified RNaseA with the polymer P1; c) Analysis of size distribution ofthe protein, polymer, and the polymer-protein complex; d) Temporalevolution fluorescence polarization of the green fluorescent protein(GFP) in the presence of the polymer with time at 2.3 nM concentrationat 1:10 ratio.

FIG. 3 . Traceless release of RNaseA from the polymer-protein complex.a) release kinetics of RNaseA protein from the RNaseA-BA@P1 complex inthe presence of 1 mM and 10 mM H₂O₂; b) circular dichroism (CD) spectraof the protein, modified protein, the polymer-protein complex, and thepolymer-protein complex in the presence of H₂O₂; c) activity assay ofRNaseA using the commercially available RNaseAlert QC system that showsthat while the protein activity is suppressed in the polymer-proteincomplex, it is recovered in the presence of H₂O₂; d) ESI-MS analysis ofthe protein released from the polymer-protein complex, indicating atraceless protein release process.

FIG. 4 . Cellular uptake of the polymer-protein conjugate andROS-responsive protein release. a) confocal microscopy image of HeLacells incubated with the polymer-protein complex for 4 h, where theprotein is labeled with a fluorophore (red color represents rhodamine blabeled RNase A, blue color represents hochest 33342 dye labelednucleus); b) temporal evolution of co-localization of lysotracker(green) and the fluorescently-labeled protein (red) indicating endosomeescape in cells (nucleus were labeled by hochest 33342 dye with bluecolor); c) cytotoxicity of RNaseA the protein by itself after 48 hincubation, which indicates that RNaseA does not have access to thecytosol of cells; d) cytotoxicity of RNaseA-BA@polymer after incubationwith 200 nM of PMA to introduce oxidative stress (after 48 h), where thedose-dependent cytotoxicity shows protein release and activity insidecells.

FIG. 5 . Protein variations in the polymer-protein conjugates and theircellular uptake. a) confocal microscopy image of HeLa cells incubatedwith BSA-BA@polymer complex for 4 h, where the protein is labeled with afluorophore; b) confocal microscopy image of HeLa cells incubated withGFP-BA@polymer complex for 4 h; c) enzyme activity assays of β-gal,β-gal-BA@polymer complex in the absence and presence of H₂O₂, whichindicate that the protein is not deactivated upon complexation; d) X-galcellular assay for β-gal protein and the β-gal-BA@polymer complex,showing that the complex is essential for the cellular uptake.

FIG. 6 . Formation of the polymer-protein conjugate with a disulfidelinker and thiol-responsive protein release. a) modification of lysinesurface functional groups in proteins with boronic acid with aself-immolative reduction-sensitive linker, where reducing agents suchas DTT and GSH would result in traceless protein release; b) SDS-PAGEgel data, illustrating the complexation and stimulus-induced disassemblyof the complex (lane 1: RNaseA-SS-BA; lane 2: complex, lane 3: complexafter incubation 10 mM concentration of GSH; lane 3: complex afterincubation 10 mM concentration of DTT); c) cytotoxicity of thereduction-sensitive RNaseA-polymer complex after incubation with HeLacells.

FIG. 7 . ¹H NMR spectrum of molecule 1.

FIG. 8 . ¹H NMR spectrum of molecule 3a.

FIG. 9 . ¹H NMR spectrum of molecule 3b.

FIG. 10 . ¹H NMR spectrum of molecule 3c.

FIG. 11 . ¹H NMR spectrum of molecule 3d.

FIG. 12 . ¹³C NMR spectrum of molecule 3d.

FIG. 13 . ¹H NMR spectrum of molecule 3e.

FIG. 14 . ¹³C NMR spectrum of molecule 3e.

FIG. 15 . ¹H NMR spectrum of molecule 3.

FIG. 16 . ¹³C NMR spectrum of molecule 3.

FIG. 17 . ¹H NMR spectrum of molecule 4.

FIG. 18 . ¹³C NMR spectrum of molecule 4.

FIG. 19 . ¹H NMR spectrum of P1′.

FIG. 20 . ¹H NMR spectra comparison for P1′ and P1. NMR solvent for P1′is Acetone-d₆ and P1 is CDCl₃.

FIG. 21 . Molecular weight information for different polymers.

FIG. 22 . ¹H NMR spectrum of molecule 6.

FIG. 23 . ¹³C NMR spectrum of molecule 6.

FIG. 24 . ¹H NMR spectrum of molecule 7a.

FIG. 25 . ¹³C NMR spectrum of molecule 7a.

FIG. 26 . ¹H NMR spectrum of molecule 7b.

FIG. 27 . ¹³C NMR spectrum of molecule 7b.

FIG. 28 . ¹H NMR spectrum of molecule 7.

FIG. 29 . ¹³C NMR spectrum of molecule 7.

FIG. 30 . MADLI-MS of RNase A before and after different sensitivelinker modification. The Mw of unmodified RNase is 13700 Da. The averageMWs for RNase A-BA, RNase A-SS-BA and RNase A-BA_pH are 15380, 17128 and15930 respectively. Based on the calculation, the average amount ofmodification for RNase A-BA, RNase A-SS-BA and RNase A-BA_pH are 9, 10and 8 respectively. FIG. 31 . Full gel for RNase A related complex. 1,RNase A; 2, RNase A-BA; 3, RNase A-BA+H₂O₂ (10 mM); 4, RNase A+polymer(1:10); 5-8, RNase A+polymer with different ratios (5, 1:0.5; 6, 1:1; 7,1:5, 8, 1:10); 9-10, complex 7+H₂O₂ (9, 1 mM; 10, 10 mM).

FIG. 32 . Fluorescence titration through ARS assay. The left figure isthe illustration of the titration process: ARS dye is complexed withboronic acid modified protein at first. Then, the polymer solution wasadded into the above complex. The result readout was recorded by thedecrease of the fluorescence as shown in the right figure.

FIG. 33 . Polymer toxicity from MTT assay toward different cell lines(HeLa, MDA-MB-231, MCF-7) for different incubation times (24 h and 48h).

FIG. 34 . Polymer toxicity from Alamar blue assay toward HeLa cell lineafter 24 h incubation.

FIG. 35 . Cell uptake of Rhodamine B labelled polymer (0.3 mg/mL) withCLSM imaging at different magnifications. (HeLa cell, 4 h incubation)

FIG. 36 . Cell uptake of RNaseA, RNaseA-BA, and RNaseA-BA@polymer at thesame protein concentration (30 μg/mL). (HeLa cell line, 4 h incubation)

FIG. 37 . Confocal imaging of cell uptake of RNaseA complex (proteinconcentration: 30 μg/mL) after 4 h incubation.

FIG. 38 . Confocal imaging of cell uptake of RNaseA complex (proteinconcentration: 30 μg/mL) after 24 h incubation.

FIG. 39 . Cytotoxicity (HeLa cell) study (MTT assay) after 48 hincubation. a) RNaseA-BA, b) RNaseA-BA@polymer.

FIG. 40 . Cytotoxicity (HeLa cell) study (MTT assay) in the presence of200 nM PMA after 48 h incubation. a) Polymer P1, b) RNaseA, c)RNaseA-BA.

FIG. 41 . Size comparison of different proteins used in this work. ThePDB codes for protein structures were-RNase A: 5rsa; GFP: 1gfl; BSA:3v03; β-gal: 1jz8.

FIG. 42 . SDS-PAGE gel for GFP complexation. Lane 1. GFP; Lane 2.GFP-BA; Lane 3. GFP-BA+H₂O₂ (10 mM), Lane 4. Polymer P1; Lane 5.GFP+polymer (1:10); Lane 6. GFP-BA+polymer (1:10); Lane 7.GFP-BA@polymer+H₂O₂ (10 mM concentration).

FIG. 43 . MALDI-MS for GFP before and after boronic acid linkermodification. The average Mw for GFP and GFP-BA are 26900 and 28793 Da.Based on the calculation, the average modification amount is 11. FIG. 44. CD spectra for GFP and related complex (including the protein,modified protein, the polymer-protein complex, and the polymer ascontrol).

FIG. 45 . Gel for BSA and related complex. Lane 1, BSA; Lane 2, BSA-BA;Lane 3, BSA-BA+H₂O₂ (1 mM concentration); Lane 4, BSA-BA@polymer; Lane5, BSA-BA@polymer+H₂O₂ (1 mM concentration).

FIG. 46 . Concentration dependent cell uptake of BSA-BA@polymer (at 1:10ratio) complex (HeLa cell line, 4 h incubation). Protein concentrations:a) 12.5 μg/mL, b) 50 μg/mL, c) 100 μg/mL. BSA was labelled withrhodamine B.

FIG. 47 . Gel for β-Gal complexation and release process. 1, β-gal; 2,β-gal-BA; 3, β-gal-BA+H₂O₂; 4, Polymer P1; 5, β-gal+Polymer; 6-9,β-gal-BA&Polymer (1:1; 1:2; 1:5; 1:10); 10, 8+H₂O₂(10 mM).

FIG. 48 . Standard curve for β-Gal activity assay.

FIG. 49 . Cell line variations in the cellular uptake usingpolymer-protein conjugates. a) confocal microscopy image of MDA-MB-231cells incubated with RNaseA-BA@polymer complex for 4 h; b) confocalmicroscopy image of MCF-7 cells incubated with RNaseA-BA@polymer complexfor 4 h.

FIG. 50 . Formation of the polymer-protein conjugate with thecis-aconityl linker and pH-responsive protein release. a) modificationof lysine surface functional groups in proteins with boronic acid withan intervening pH-sensitive linker, where lowering the pH would resultin traceless protein release; b) SDS-PAGE gel data, illustrating thecomplexation and stimulus-induced disassembly of the complex (lane 1:RNase A-BA_pH; lane 2: complex, lane 3: complex @ acidic condition); c)cytotoxicity of RNaseA-BA with the pH sensitive linker attached to theprotein (after 48 h incubation), which indicates that RNaseA-BA with pHsensitive linker by itself does not have access to the cytosol of cells;d) cytotoxicity of the pH-sensitive RNaseA-polymer complex afterincubation with HeLa cells.

FIG. 51 . Gel for redox responsiveness of RNase A and related complex.Lane 1, RNase A; Lane 2, RNase A-SS-BA; Lane 3, RNase A-SS-BA+GSH (10mM); Lane 4, RNase A+polymer (1:10); Lane 5, RNase A-SS-BA+polymer(1:10); Lane 6, 5+GSH (10 mM); Lane 7, 5+DTT (10 mM); Lane 8, 5+H₂O₂(10mM); Lane 9, 5+Glucose (2 mg/mL).

FIG. 52 . Cytotoxicity of RNase A-SS-BA after 48 h incubation (HeLacell, MTT assay).

FIG. 53 . Confocal images for cell uptake based on different polymers.

FIG. 54 . Flow cytometry for quantification of cell uptake based ondifferent polymers.

FIG. 55 . Structure of the molecule 1, involved in the modification ofsurface lysines in proteins, structure of the polymer P1, used for theclick-induced formation of the polymer-protein complex. This complex canthen be degraded to afford the protein, without any remnants of thepolymer in the presence of a reactive oxygen species stimulus.

FIG. 56 . Structures of certain exemplified polymers. FIG. 57 .Structures of polymer structures P3′-P12′.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an efficient and effective deliveryplatform for intracellular delivery of proteins. The protein deliverysystem disclosed herein is a simple click chemistry approach, a simpleand rapid ‘mix and go’ approach, that offers rapid and reversibleconjugation capabilities, which are typical to non-covalentinteractions, combined with the robust conjugate stability, a keycharacteristic of covalent methods.

As illustrated in FIG. 1 , the protein delivery approach has thepotential to present a non-fouling surface with robust conjugatestability characteristics offered by the covalent methods.

Construction of polymer-protein nanoassemblies is a challenge asreactions between macromolecules, especially those involving proteins,are inherently inefficient owing to the sparse reactive functionalgroups and low concentration requirements. This challenge was addressedherein using an ultrafast and reversible click reaction, which forms thebasis for a covalent self-assembly strategy between side-chainfunctionalized polymers and surface-modified proteins.

Linkers embedded in the molecular assembly have been programmed torelease the incarcerated proteins in its native form, only whensubjected to the presence of a specific trigger. The generality and theversatility of the approach are herein demonstrated by showing that thedisclosed approach can be used for proteins of different sizes andisoelectric points.

Surface modified proteins can be rapidly conjugated with polymers, whichcan be fully reversed in the presence of a specific and biologicallyrelevant stimulus. The broad applicability of the molecular designstrategy has been illustrated with encapsulations of proteins ofdifferent sizes and isoelectric points (pI) and the release ofencapsulated proteins in response to three different stimuli, viz.reactive oxygen species (ROS), reducing environment, and variations inpH. In addition to the encapsulation and release of proteins, theutility of this self-assembly process is also demonstrated withintracellular delivery of these proteins with retention of function.

Modifications in the linker chemistry offers the ability to triggerthese assemblies with various chemical inputs. Efficient formation ofnanoassemblies based on polymer-protein conjugates has implications in avariety of areas at the interface of chemistry with materials andbiology, such as in the generation of active surfaces and in delivery ofbiologics.

In one aspect, the invention generally relates to a functionalizedcopolymer, comprising: a first monomer of PEG-methacrylate (PEG-MA); anda second monomer of methacrylate having a side chain modified with asalicylhydroxamate moiety.

In certain embodiments of the functionalized copolymer, the firstmonomer is the majority monomer and the second monomer is the minoritymonomer.

In certain embodiments, the first monomer comprises a side chaincomprising from about 1 to about 20 (e.g., from about 1 to about 15,from about 1 to about 10, from about 1 to about 5, from about 5 to about20, from about 10 to about 20, from about 5 to about 15, from about 5 toabout 10) ethylene-oxide units. In certain embodiments, the firstmonomer has the structure of:

where m is an integer in the range of 1 to 20 (e.g., from about 1 toabout 15, from about 1 to about 10, from about 1 to about 5, from about5 to about 20, from about 10 to about 20, from about 5 to about 15, fromabout 5 to about 10).

In certain embodiments, the second monomer comprises:

or a protected form thereof.

In certain embodiments, the second monomer has the structure of:

or a protected form thereof, where n is an integer in the range of 1 to20 (e.g., from about 1 to about 15, from about 1 to about 10, from about1 to about 5, from about 5 to about 20, from about 10 to about 20, fromabout 5 to about 15, from about 5 to about 10).

In certain embodiments, the functionalized copolymer has the structuralformula:

wherein each of m and n is independently an integer in the range of 1 to20 (e.g., from about 1 to about 15, from about 1 to about 10, from about1 to about 5, from about 5 to about 20, from about 10 to about 20, fromabout 5 to about 15, from about 5 to about 10), and x:y is in the rangefrom about 5:95 to about 70:30 (e.g., from about 5:95 to about 60:40,from about 5:95 to about 50:50, from about 10:90 to about 70:30, fromabout 20:80 to about 70:30, from about 30:70 to about 70:30, from about40:60 to about 70:30, from about 50:50 to about 70:30, about 1:1).

In certain embodiments, the functionalized copolymer has a molecularweight (M_(w)) in the range of about 1 k to about 200 k (e.g., about 1 kto about 100 k, about 1 k to about 50 k, about 1 k to about 20 k, about1 k to about 10 k, about 5 k to about 200 k, about 10 k to about 200 k,about 20 k to about 200 k, about 50 k to about 200 k, about 5 k to about50 k, about 5 k to about 20 k).

In another aspect, the invention generally relates to a surface modifiedprotein comprising arylboronic acid modifications of one or more lysineresidues.

In certain embodiments of the surface modified protein, the arylboronicacid is a phenylboronic acid.

In certain embodiments, one or more lysine residues of the protein ismodified by

wherein n is an integer in the range of 1 to 20 (e.g., from about 1 toabout 15, from about 1 to about 10, from about 1 to about 5, from about5 to about 20, from about 10 to about 20, from about 5 to about 15).

In certain embodiments, the arylboronic acid modification is conjugatedto the protein via a degradable linker sensitive to reactive oxygenspecies, a reducing environment, or change in pH.

In certain embodiments, the reactive oxygen species is hydrogenperoxide. In certain embodiments, the reducing environment is offered byhigher intracellular glutathione (GSH) concentrations. In certainembodiments, change in pH is caused by a lower pH of cellularmicroenvironments (e.g., in cancer cells and in endosomal/lysosomalcompartments).

In certain embodiments, the arylboronic acid modification is conjugatedto the protein via a boronate ester linker. In certain embodiments, thearylboronic acid modification is conjugated to the protein via adisulfide linker. In certain embodiments, the arylboronic acidmodification is conjugated to the protein via a cis-aconityl linker.

In certain embodiments, the protein has a molecular weight in the rangeof about 10 k to about 500 k (e.g., about 20 k to about 500 k, about 50k to about 500 k, about 100 k to about 500 k, about 10 k to about 100 k,about 10 k to about 50 k).

In certain embodiments, the protein has an isoelectric points (pI) inthe range of about 3.0 to about 12.0 (e.g., about 5.0 to about 12.0,about 7.0 to about 12.0, about 9.0 to about 12.0, about 3.0 to about9.0, about 3.0 to about 7.0, about 3.0 to about 5.0, about 5.0 to about10.0).

In yet another aspect, the invention generally relates to apolymer-protein conjugate, comprising: a copolymer comprising a firstmonomer of PEG-methacrylate (PEG-MA) and a second monomer ofmethacrylate having a side chain conjugated to a protein via adegradable linker.

In certain embodiments of the polymer-protein conjugate, wherein thedegradable linker is sensitive to reactive oxygen species, a reducingenvironment, or change in pH. In certain embodiments, the reactiveoxygen species is hydrogen peroxide (H₂O₂). In certain embodiments, thereducing environment is offered by higher intracellular glutathioneconcentrations. In certain embodiments, change in pH is caused by alower pH of cellular microenvironments (e.g., in cancer cells and inendosomal/lysosomal compartments).

In certain embodiments, the degradable linker comprises a boronate esterlinker. In certain embodiments, the degradable linker comprises adisulfide linker. In certain embodiments, the degradable linkercomprises a cis-aconityl linker.

In yet another aspect, the invention generally relates to a molecularassembly comprising the polymer-protein conjugate disclosed herein.

In certain embodiments of the molecular assembly, the polymer:proteinratio by weight is in the range from about 1:1 to about 50:1 (e.g., fromabout 1:1 to about 30:1, from about 1:1 to about 20:1, from about 1:1 toabout 10:1, from about 1:1 to about 5:1, from about 1:1 to about 3:1,from about 5:1 to about 50:1, from about 10:1 to about 50:1, from about20:1 to about 50:1, from about 2:1 to about 10:1, from about 2:1 toabout 8:1, from about 2:1 to about 5:1, from about 5:1 to about 10:1).

In certain embodiments, the polymer-protein conjugate is adapted torelease the protein in its native form upon degradation of thedegradable linker.

In certain embodiments, the degradable linker comprises a boronate esterlinker. In certain embodiments, the degradable linker comprises adisulfide linker. In certain embodiments, the degradable linkercomprises a cis-aconityl linker.

In certain embodiments, the polymer-protein conjugate is adapted torelease the protein in the presence of a specific and biologicallyrelevant stimulus inside cells. In certain embodiments, thepolymer-protein conjugate is adapted to release the protein in thepresence of a specific and biologically relevant stimulus in thecytosol.

In certain embodiments, the protein has a molecular weight in the rangeof about 10 k to about 500 k (e.g., about 20 k to about 500 k, about 50k to about 500 k, about 100 k to about 500 k, about 10 k to about 100 k,about 10 k to about 50 k).

In certain embodiments, the protein has an isoelectric point (pI) in therange of about 3.0 to about 12.0 (e.g., about 5.0 to about 12.0, about7.0 to about 12.0, about 9.0 to about 12.0, about 3.0 to about 9.0,about 3.0 to about 7.0, about 3.0 to about 5.0, about 5.0 to about10.0).

In yet another aspect, the invention generally relates to a compositioncomprising the molecular assembly disclosed herein.

In yet another aspect, the invention generally relates to a method fordelivering a protein, comprising: surface functionalizing the proteinwith arylboronic acid modifications of one or more lysine residues onthe protein; forming a polymer-protein conjugate by reacting the surfacefunctionalized protein with a copolymer comprising a first monomer ofPEG-methacrylate (PEG-MA) and a second monomer of methacrylate having aside chain modified by a salicylhydroxamate moiety thereby forming amolecular assembly comprising the polymer-protein conjugate, wherein thepolymer-protein conjugate comprises a degradable linker; transportingthe molecular assembly to a target site to degrade the linker (and thusthe molecular assembly) thereby releasing the protein at the targetsite.

In certain embodiments of the method, the target site is inside a cell.In certain embodiments, the protein is released for its native functionin the presence of a specific and biologically relevant stimulus insidecells.

In certain embodiments, the target site is cytosol. In certainembodiments, the polymer-protein conjugate is adapted to release theprotein in the presence of a specific and biologically relevant stimulusin the cytosol.

In certain embodiments, the specific and biologically relevant stimulusare reactive oxygen species (e.g., hydrogen peroxide). In certainembodiments, the specific and biologically relevant stimulus is areducing environment offered by higher intracellular glutathioneconcentrations. In certain embodiments, the specific and biologicallyrelevant stimulus is a change in pH is caused by a lower pH of cellularmicroenvironments (e.g., in cancer cells and in endosomal/lysosomalcompartments).

In certain embodiments, the polymer:protein ratio by weight is in therange from about 1:1 to about 50:1 (e.g., from about 1:1 to about 30:1,from about 1:1 to about 20:1, from about 1:1 to about 10:1, from about1:1 to about 5:1, from about 1:1 to about 3:1, from about 5:1 to about50:1, from about 10:1 to about 50:1, from about 20:1 to about 50:1, fromabout 2:1 to about 10:1, from about 2:1 to about 8:1, from about 2:1 toabout 5:1, from about 5:1 to about 10:1).

In certain embodiments, the protein is released in its native form forits native function. In certain embodiments, the released proteinretains at least 70% (e.g., at least 80%, at least 90%, at least 95%, atleast 99%) of the activity of the native protein (i.e., the protein'snative function).

The term “protein” as used herein refers to a polymer of amino acidresidues (a “polypeptide”) and is not limited to a minimum length. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also includepost-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation, and the like. Furthermore,a polypeptide may refer to a protein which includes modifications, suchas deletions, additions, and substitutions (generally conservative innature), to the native sequence, as long as the protein maintains thedesired activity. These modifications may be deliberate or may beaccidental. Amino acids can be referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.

In certain embodiments, wherein the degradable linker comprises aboronate ester linker. In certain embodiments, the degradable linkercomprises a disulfide linker. In certain embodiments, the degradablelinker comprises a cis-aconityl linker.

In certain embodiments, the protein has a molecular weight in the rangeof about 10 k to about 500 k (e.g., about 20 k to about 500 k, about 50k to about 500 k, about 100 k to about 500 k, about 10 k to about 100 k,about 10 k to about 50 k).

In certain embodiments, the protein has an isoelectric points (pI) inthe range of about 3.0 to about 12.0 (e.g., about 5.0 to about 12.0,about 7.0 to about 12.0, about 9.0 to about 12.0, about 3.0 to about9.0, about 3.0 to about 7.0, about 3.0 to about 5.0, about 5.0 to about10.0).

In yet another aspect, the invention generally relates to a method forforming a molecular assembly, comprising: surface functionalizing theprotein with arylboronic acid modifications of one or more lysineresidues on the protein; and reacting the surface functionalized proteinwith a copolymer comprising a first monomer of PEG-methacrylate (PEG-MA)and a second monomer of methacrylate having a side chain modified by asalicylhydroxamate moiety thereby forming a molecular assemblycomprising the polymer-protein conjugate.

In certain embodiments of the method, wherein the first monomer of thecopolymer comprises a side chain comprising from about 1 to about 20(e.g., from about 1 to about 15, from about 1 to about 10, from about 1to about 5, from about 5 to about 20, from about 10 to about 20, fromabout 5 to about 15) ethylene-oxide units.

In certain embodiments, the first monomer has the structure of:

where m is an integer in the range of 1 to 20 (e.g., from about 1 toabout 15, from about 1 to about 10, from about 1 to about 5, from about5 to about 20, from about 10 to about 20, from about 5 to about 15).

In certain embodiments, the second monomer of the copolymer comprises:

or a protected form thereof.

In certain embodiments, the second monomer has the structure of:

or a protected form thereof, where n is an integer in the range of 1 to20 (e.g., from about 1 to about 15, from about 1 to about 10, from about1 to about 5, from about 5 to about 20, from about 10 to about 20, fromabout 5 to about 15).

In certain embodiments, the copolymer has the structural formula:

wherein each of m and n is independently an integer in the range of 1 to20 (e.g., from about 1 to about 15, from about 1 to about 10, from about1 to about 5, from about 5 to about 20, from about 10 to about 20, fromabout 5 to about 15), and x:y is in the range from about 5:95 to about70:30 (e.g., from about 5:95 to about 60:40, from about 5:95 to about50:50, from about 10:90 to about 70:30, from about 20:80 to about 70:30,from about 30:70 to about 70:30, from about 40:60 to about 70:30, fromabout 50:50 to about 70:30, about 1:1).

In certain embodiments, the copolymer has a molecular weight (M_(w)) inthe range of about 1 k to about 200 k (e.g., about 1 k to about 50 k,about 1 k to about 20 k, about 1 k to about 10 k, about 5 k to about 200k, about 10 k to about 200 k, about 20 k to about 200 k, about 50 k toabout 200 k, about 5 k to about 100 k, about 5 k to about 50 k).

In certain embodiments, one or more lysine residues of the protein ismodified by

wherein n is an integer in the range of 1 to 20 (e.g., from about 1 toabout 15, from about 1 to about 10, from about 1 to about 5, from about5 to about 20, from about 10 to about 20, from about 5 to about 15).

EXAMPLES

Salicylhydroxamate-arylboronic acid combination was chosen as the clickreaction of choice, because of its high efficiency with a typicalreaction rate of 7×10 6 M⁻²s⁻¹. (Arzt, et al. Chem. Asian J. 2014, 9,1994-2003; Ng, et al. Angew. Chem. Int. Ed. 2014, 53, 324-328; Shin, etal. Chem. Biol. 2010, 17, 1171-1176.)

Neither of these functional groups, however, are natively present inproteins. Therefore, in order to use this click reaction as the key stepin the polymer-protein conjugate formation, one of these functionalgroups must be installed on the protein surface. To insure broadapplicability of this approach, it is also critical that an amino acidside chain functionality is chosen that is ubiquitous on proteinsurfaces. Thus, lysines were chosen as the functional handles to carryout the modification, as >80% of globular proteins have more than onelysine residue on their surface. Arylboronic acid was reportedly used tomodify lysines. (Wong, et al. Adv. Drug Delivery Rev. 2012, 64,1031-1045; Roth, et al. Chem. Rev. 2016, 116, 1309-1352; Blencowe, etal. Polym. Chem. 2011, 2, 773-790.)

Scheme 1 (FIG. 55 ) shows the molecular design of the disclosedapproach, where lysines on protein surfaces are treated with thep-nitrophenylcarbonate of 4-hydroxymethyl-phenylboronate ester 1. Theresultant lysine modification is hydrolytically stable. However, whenthe boronic acid moiety is converted to the corresponding phenol underoxidizing conditions, this functionality rapidly degrades back to theamine. This hydrogen peroxide induced reversibility of the lysinemodification forms the basis for the traceless release of the proteinsunder ROS conditions.

To conjugate the polymer to the protein, the salicylhydroxamate moietywas installed on to a polymer. The targeted polymer structure is shownas P1 in Scheme 1. P1 contains a high percentage of polyethyleneglycol(PEG) functionalities (90%), because of a PEG-methacrylate (PEG-MA) isused as a comonomer. PEG-MA is used as a majority co-monomer in P1, asthis should endow the polymer with water solubility and charge-neutralpolymer-protein nanoassembly with non-fouling characteristics.

To achieve a salicylhydroxamate-based monomer, selectivefunctionalization of the phenolic group at the para-position of2,4-dihydroxybenzoic acid (2) was carried out to allow the installationof a polymerizable methacrylate moiety (molecule 3, Scheme 2). Theprecursor monomer in its trityl protected form, 4, was achieved bycoupling the carboxylic acid moiety of 3 with protected hydroxylamine.The resultant monomer 4 was then copolymerized with PEG-MA (5) in 1:9ratio to obtain the salicylhydroxamate functionalized polymer P1, asshown in Scheme 2. The polymerization was carried out underreversible-addition-fragmentation-chain-transfer (RAFT) reactionconditions.

Characterization of the polymer with ¹H NMR revealed that the ratio ofthe two monomers correlated well with the feed ratio in the reaction.Gel permeation chromatography (GPC) characterization showed that theapparent molecular weight of the polymer is ˜16 kDa with thepolydispersity (Ð) of ˜1.1. This medium sized polymer was chosen forefficient reaction between polymer and protein to preparenanoassemblies. Finally, the salicylhydroxamate group was liberated bydeprotection of the trityl group using trifluoroacetic acid.

The fast reaction between the salicylhydroxamate moiety side chain inthe polymer and the boronic acid surface functionalities in the proteinshould result in a rapid self-assembly to produce a nanoassembly, asillustrated in Scheme 1. To test this possibility, ribonuclease A(RNaseA, from bovine pancreas) was used as the model protein, which waschosen for its well-established fluorescence assay for enzymaticactivity and apoptotic cellular activity. (Raines, et al. Chem. Rev.1998, 98, 1045-1066.)

Treatment of RNaseA with 1 resulted in ˜9 of the total of 11 lysineunits on the protein surface to be modified to the correspondingarylboronic acid moiety, as estimated by MALDI-MS (FIG. 30 ).

The targeted protein-polymer complex was prepared by simply mixing thepolymer P1 with the arylboronic acid-modified protein in water or PBSbuffer. The product of this self-assembly process was characterizedusing a variety of techniques. First, dynamic light scattering (DLS)studies revealed that the solution size of the complex is ˜20 nm, whichis significantly higher than any of the control samples, i.e. themodified protein, unmodified protein, polymer P1, and the physicalmixture of unmodified protein and P1 (FIG. 2 c ). Gel electrophoresis(SDS-PAGE) of the protein and the boronic acid modified protein showed awell-defined narrow band, as anticipated (FIGS. 2 a and 31).

On the other hand, RNaseA-BA@polymer complex showed a relatively smearedband at the molecular weight range, which is substantially higher thanthat of both the polymer and the protein (FIG. 2 a ). Similarly, aphysical mixture of the polymer and the unmodified protein did not causethe formation of any new band (FIG. 31 ).

These results indicate that the mixture of modified protein and thepolymer results in the formation of a higher molecular weight proteincomplex, likely due to the rapid click reaction between thesalicylhydroxamate and the aryl boronic acid moieties.

The complexation kinetics was evaluated using SDS-PAGE. The reactionbetween the polymer and the protein seemed to be complete even in 10minutes with small remnants of unreacted protein (FIG. 2 b ). In underone hour, no discernible free protein could be observed in the gel.These data show that the complexation process is quantitative and fast,even at μM concentrations of the protein.

In order to further evaluate whether this reaction can be carried out ateven lower concentrations, fluorescence was used as the probe toevaluate the conversion of the protein to the polymer-proteinnanoassembly. To this end, green fluorescent protein (GFP, recombinantprotein originated from Aequorea Victoria) was used as a model protein,as its intrinsic fluorescence can be utilized to monitor thecomplexation process with fluorescent polarization measurements.Syntheses and characterization of the arylboronic acid-modified GFP andGFP-BA@P1 complex are detailed in the SI. The fluorescent polarizationof the GFP and GFP-BA, by themselves, did not show any change with time.On the other hand, as the presumed complex between P1 and GFP-BA shouldresult in a nanoassembly with substantially higher molecular weight, thefluorescent polarization of the complexed GFP should be much higher.

Indeed, it was observed that there is a temporal evolution of thepolarization, where there is a sharp increase at ˜30 minutes and wascomplete in ˜70 min (FIG. 2 d ). The reason for the lag time of 30minutes is not clear to us at this time. It should be noted that the nMconcentration of the protein in this experiment is substantially lowerthan the experiments above, which were characterized by SDS-PAGE. Toadditionally confirm that the observed increase in polarization isindeed due to the click reaction between the polymer and the protein,the physical mixture of the unmodified GFP and P1 were evaluated, whichgratifyingly did not exhibit any change in fluorescence polarizationwith time (FIG. 2 d ).

The kinetics experiments above were carried out at the polymer:proteinratio of 10:1. The ideal ratio at which this reaction would be completewas identified. SDS-PAGE of the polymer-protein complexes at differentratios are shown in FIG. 2 a . Understandably, the extent of freeprotein band decreased with increase in the polymer:protein ratio. At5:1 ratio, there was no discernible free protein in the gel, indicatingquantitative complex formation at this ratio. Since the protein iscompletely encapsulated, no further purification might be needed toremove the free protein. To be more quantitative about the extent ofcomplexation between the polymer and the protein, the boronic acidmodified RNaseA was treated with alizarin red S (ARS) dye, which hasbeen previously used for quantifying boronic acid functionalities.(Springsteen, et al. Chem. Commun. 2001, 1608-1609.)

ARS contains a catechol moiety that complexes to the boronic acid on theprotein surface. Then, the extent of complexation between thesalicylhydroxamate polymer P1 and these modified proteins can bequantified, since the displacement of the catechol moiety in ARS withthe salicylhydroxamate moiety would cause the liberation of the dye fromthe boronic acid, which can be measured by fluorescence (FIG. 32 ). Itwas found that the reaction was complete at the polymer:protein ratio of˜3, as any further increase in the ratio did not produce any change inARS fluorescence.

Next, the possibility of ROS-responsive release of the encapsulatedprotein was assessed. Specifically, hydrogen peroxide (H₂O₂) was used asthe stimulus, as this oxidant has been implicated in many ROS-producingdiseases. (Stone, et al. Antioxid. Redox Signaling 2006, 8, 243-270;Finkel, et al. Nature 2007, 448, 767-774.)

The basis for the disclosed molecular design for traceless proteinrelease is that in the presence of H₂O₂, the boronate esters in thecomplex would be oxidized to the corresponding phenol, which would causethe protein surface to be disconnected from the polymer. Thep-hydroxybenzylcarbamate moiety would then rapidly self-immolate througha 1,6-benzyl elimination to liberate the lysines on the surface of theprotein as illustrated in Scheme 1. (Wang, et al. Angew. Chem., Int. Ed.2014, 53, 13444-13448; Jourden, et al. Angew. Chem. Int. Ed. 2010, 49,6795-6797; Lux, et al. J. Am. Chem. Soc. 2012, 134, 15758-15764;Broaders, et al. J. Am. Chem. Soc. 2011, 133, 756-758.)

This possibility of protein release was studied under two H₂O₂concentrations (1 mM and 10 mM). The temporal release of the protein wasstudied over a 24 hours period using gel electrophoresis, as shown inFIG. 3 a . Indeed, in both cases the protein release was ascertained bygradual increase in the intensity of the band that corresponds to thefree protein. The release kinetics was also found to be dependent on theconcentration of the stimulus. Also noted was that the oxidationconditions result in protein dimerization. Further, it was found thatthe protein release kinetics was independent on the polymer length, asdiscerned from testing polymer P3 with Mw of 35 kDa for theself-assembly process (FIG. 33 ).

It is understood that while the SDS-PAGE studies show that the proteinis released, this assay does not provide information on the post-releasestructure or the function of the protein. Structure of the releasedprotein was first analyzed using circular dichroism (CD). Indeed, theintensity and shape of the CD spectrum of the released protein closelymatched with that of the pristine RNaseA (FIG. 3 b ). A small, butdiscernible, shift in the band at 210 nm was noticed. While this can beattributed to the fact that the released protein contains the polymer,byproducts of the self-immolation reaction, and the oxidant, of interestwas testing the fidelity of the process by testing the activity of thereleased protein. Thus, the released protein was subjected to awell-established fluorogenic assay for RNaseA activity. The releasedprotein retained >90% activity of the pristine protein (FIG. 3 c ). Tofurther confirm that the protein is indeed released in its tracelessform, the released proteins were analyzed using mass spectrometry, as itis sensitive to subtle changes in mass and thus would be the clearestindicator of traceless release. Indeed, the released protein exhibitedthe same mass as the pristine RNase A (FIG. 3 d ).

One of the goals here is to utilize this self-assembly approach totraffic globular proteins across the cellular membrane, as the use ofthe polymer sheath to mask the proteins, gain access to the cellularinterior, and tracelessly release the protein cargo inside the cytosolin its functionally active form. Prior to testing this possibility, itwas needed to test whether the polymer exhibits any cytotoxicity. Tothis end, the toxicity of the polymer P1 was tested using the Alamarblue assay and MTT assay; the polymer P1 did not exhibit any toxicitytoward different cell lines (HeLa, MDA-MB-231, MCF-7) after differentincubation times, even at 1.0 mg/mL concentration (FIGS. 34 and 35 ). Toensure that the polymer is indeed gaining access into the cells and isstill non-toxic, the polymer was labeled with a small percentage (˜1%)of a fluorophore, rhodamine B, to generate polymer P2. When the cellswere incubated with this labeled polymer for the same amount of time,the cellular uptake was found to be significantly high (FIG. 36 ).Overall, these results show that the polymer itself is non-toxic andthus can be used for intracellular protein delivery experiments.

The possibility of intracellular delivery of proteins was tested usingRNaseA as the protein in HeLa cells. Since the focus was mainly on thelocation of the protein in the experiments here, RNase A-BA wasfluorescently labeled with rhodamine B. Upon incubating thepolymer-protein conjugate with cells, the intracellular uptake wastracked using confocal laser scanning microscopy (CLSM) at differenttimes. Concurrently, the nucleus of the cells was labeled with Hoechst33342. After just 4 h, a well-distributed fluorescence from the labeledproteins was observed (FIG. 4 a and FIG. 37 ), while negligiblefluorescence was observed from cells that were treated with an identicalconcentration of naked proteins (FIG. 37 ). Interestingly, RNaseA-BAitself did exhibit some uptake, which is attributed to the possibilitythat boronic acid moieties can interact with the cell membrane tofacilitate cellular uptake. (Ellis, et al. J. Am. Chem. Soc. 2012, 134,3631-3634.) Nonetheless, the efficiency of this process was much lowerthan that of the RNaseA@P1 complex.

It is also important to test whether the protein is in the cytosol.Because most nanoassemblies are thought to access cells through anendocytosis process, which means that the nanoassembly needs to escapethe endosome in order to access the cytosol. Note that RNaseA iseffective in its function, only in the cytosol. Therefore, the endosomeswere incubated with lysotracker green (FIG. 38-39 ). The initialcolocalization of green and red fluorescence did indicate that thenanoassemblies enter the cells through the endosomes, as indicated bythe fluorescence image at the 4 h timeframe (FIG. 4 b and FIG. 38 ).Interestingly however, the proteins do escape the endosomes over time,as seen by the distinct red color in the cells after 24 h (FIG. 4 b andFIG. 39 ). The mechanism for endosomal escape is not clear to us at thistime.

If the protein cargo does indeed end up in the cytosol, then thedelivered RNaseA should be functional inside the cells. RNaseA, cleavesRNA molecules inside cells to induce apoptosis. This possibility ofcellular apoptosis, after incubation with protein-containingnanoassemblies, was studied. Interestingly, none of the RNaseA-basedformulations (bare RNaseA, RNaseA-BA and RNaseA-BA@polymer) exhibitedany sign of cellular apoptosis (FIG. 4 and FIG. 40 ). This could bebecause the conjugation of the protein with the polymer causes theformer to lose its activity and that the protein has not been liberatedfrom the polymer inside the cells. This is understandable, because thesenanoassemblies are programmed to liberate the protein in the presence ofH₂O₂ as the ROS stimulus and the native ROS concentration of the HeLacell might not be sufficient. To test this idea, cells with 200 nMconcentration of phorbol-12-myristate-13-acetate (PMA) were incubated,as this molecule is known to induce ROS generation inside cells.(Kuwabara, et al. PLoS One 2015, 10, e0116410.)

Now, the RNaseA-BA@P1 exhibited a clear dosage-dependent cellulartoxicity, indicating that the protein is indeed causing cellularapoptosis. To further check that this is a manifestation of thepolymer-protein conjugate, the cells were incubated with RNaseA,RNaseA-BA, and the polymer (each by itself) in the presence of 200 nMPMA and none of these combinations exhibited any cellular toxicity (FIG.41 ).

Following the demonstration of the polymer-protein self-assembly,stimulus-induced protein release in its traceless form, and theretention of protein function including in cells, the versatility andgenerality of the approach were tested even further. To this end, thepotential was tested for delivering proteins of different sizes and todifferent cell lines. Also of interest was testing the generality of themolecular design for responsive release with other stimuli.

RNase A is a small protein with MW of 14 kDa and an isoelectric point(pI) of 8.9. The latter feature indicates that the protein is basic,i.e., there is a relatively high number of lysine units, which were usedas the handle for conjugating the proteins with the polymer. To furthertest the scope of this approach, the proteins chosen were not only ofdifferent sizes, but also that are in general considered to be moreacidic (Table 1 and FIG. 42 ). Thus, three other proteins were chosen,viz., green fluorescent protein (GFP, 27 kDa, pI 5.7), bovine serumalbumin (BSA, 66 kDa, pI 5.8) and β-galactosidase (β-gal, 484 kDa(tetramer MW), pI 5.3, from Escherichia coli). Similar to the strategyused with RNaseA, the surface lysines of these proteins were initiallymodified with 1 to convert these to the corresponding boronic acidmoieties. These modified proteins were then treated with P1, as withRNaseA-BA, to obtain the polymer-protein conjugate (FIG. 43-49 ).

TABLE 1 Protein information summary Protein M_(n) Lys (#) PI RNaseA~13,700 (150 AA) 11 8.93 GFP   26,900 (238 AA) 20 5.67 BSA ~66,000 (607AA) 60 5.82 Beta-gal    116,483 (1,024 AA) 20 5.28 464,000(homo-tetramer)

These conjugates were initially characterized for their cellular uptake.The inherent fluorescence of GFP was used as the cellular uptakereadout, while BSA was labeled with rhodamine B to track thepolymer-protein conjugate in cells. As shown in FIG. 5 , both GFP-BA@P1and BSA-BA@P1 exhibited excellent cellular uptake. Since both theseproteins do not have a functional assay, the fate of the polymer-proteinconjugate further inside the cells was not probed. On the other hand,β-gal activity can be readily assessed with the X-gal assay.

Interestingly, the polymer conjugation did not result in any loss ofβ-gal function (FIG. 5 c ). Note that while the conjugation of thepolymer to the protein could result in loss of activity, as with RNaseA,possibly due to restricted protein mobility, it is not obvious that allproteins would lose activity because of the conjugation process.Therefore, it is not surprising that β-gal did not lose its activityupon conjugation. This feature also allowed us to probe a possibility,when delivering these conjugates inside the cells; i.e., thispolymer-protein should not require activation by H₂O₂ in order toexhibit its activity. However, the fact that the protein is conjugatedto the polymer should allow it to be taken up by the cells.

To test this idea, β-gal-BA@P1 was incubated with HeLa cells and assayedthe activity of β-gal after 24 hours. The blue color of the cells showedthat there is significant β-gal activity in the cells. In the controlexperiment, where the cells were incubated with the β-gal protein byitself, there was no discernible activity of the enzyme inside the cells(FIG. 5 d ). Overall, these experiments indicate that the disclosedprotein conjugation and delivery strategy is broadly applicable toproteins of different sizes.

Similarly, cellular uptake experiments were carried out using labeledproteins in two other cancer cell lines, viz. MDA-MB-231 and MCF-7. Thecellular uptake was found to be very high in both these cases (FIG. 50), indicating that the strategy is applicable to other cell lines aswell. Note also that these cellular uptake studies are being carried outwithout the incorporation of any surface ligand, relying on passiveuptake. Opportunities do exist for cellular targeting through ligandincorporation for activated cellular uptake.

Since the salicylhydroxamate-boronic acid-based conjugate offers theopportunity for protein release only in the presence of ROS stimuluseven inside cells, of interest was exploring the potential for expandingthis approach to other stimuli, while still taking advantage of the veryfast click reaction kinetics. Two other stimuli were targeted for thispurpose; to utilize the inherently reducing conditions of the cytosoland to utilize the lower pH of cellular microenvironments such as incancer cells and in endosomal/lysosomal compartments. (Ventura, et al.Biomacromolecules 2015, 16, 3161-3171; Liu, et al. J. Am. Chem. Soc.2017, 139, 2306-2317; Casey, et al. Nat. Rev. Mol. Cell Biol. 2010, 11,50-61; Webb, et al. Nat. Rev. Cancer 2011, 11, 671-677.)

In both of these cases, an appropriate functional group was introducedbetween the lysine moiety of the protein and the boronic acidfunctionality such that the specific stimulus-induced degradation of afunctional group would result in the regeneration of the lysine unit.

First focused was on developing a system that would release the proteinin response to lower pH at specific microenvironments. The pH-sensitivecis-aconityl linker was introduced between the lysine and the boronicacid moiety. (Lee, et al. Angew. Chem., Int. Ed. 2009, 48, 5309-5312.)

Accordingly, RNaseA lysines were modified with the linker 6, shown inFIG. 51 . Following the modification, as with the methods outlinedabove, the protein was treated with P1 to generate the conjugate. Whenthis conjugate was subjected to lower pH for a period of time, theprotein was liberated from the nanoassembly as discerned by the SDS-PAGEstudies. Similar to the ROS-induced release studies above, thisconjugate was also incubated with cells. Compared to the controls, theconjugate indeed exhibited cellular apoptosis; but, the cellulartoxicity here was found to be lower than that observed with theROS-responsive system. This is understandable, because the protein inthis case is likely to be partially or fully cleaved from the polymer inthe endosome. The extent of endosomal escape for the protein by itselfmight be low, which is attributed to the lower cell kill.

Next targeted was a system that would respond to the reducingenvironment of the cells, where the linker would be processed in thecytosol instead of the endosome. To be responsive to the reducingenvironment, offered by higher intracellular glutathione concentrations,a disulfide linker was introduced at the (β-position relative to thecarbamate moiety. Reduction based cleavage and subsequent generation ofa thiol moiety causes an intramolecular nucleophilic attack on thecarbamate moiety to liberate the lysine unit. (Riber, et al. Adv.Healthcare Mater. 2015, 4, 1887-1890.)

A molecular design that offers the possibility of reduction-inducedtraceless release of proteins in this click chemistry based conjugatesystem is shown as 7 in FIG. 6 . Treatment of the modified protein,RNaseA-SS-BA, with P1 affords the corresponding polymer-proteinconjugate RNaseA-SS-BA@Pl. The possibility of releasing the protein inthe presence of physiologically relevant, intracellular concentration ofGSH and the corresponding concentration of DTT was analyzed usingSDS-PAGE.

Indeed, significant protein release was observed in the presence ofthese reducing agents, as shown FIG. 6 and FIG. 52 . The true test ofthe fidelity of these assemblies is to test whether the functional formof protein can be delivered inside the cells, where the high nativeconcentration of GSH would degrade the polymer-protein conjugate tocause cellular apoptosis. Indeed, a dosage-dependent cellular toxicitywas noted. Since the experiment here, the PMA-induced degradationexperiments, and the pH-responsive system were all carried out withrespect to protein concentrations, the cellular apoptosis arecomparable. It can be noted here that the reduction induced proteinrelease is not quite as efficient as the ROS-responsive system, but isunderstandably better than the pH-responsive system. This is attributedto the fact that the nanoassembly is processed in the cytosol, but thereis need for the diffusion of a larger GSH molecule inside the nanogelsto cleave the disulfide bonds, relative to the smaller H₂O₂ molecules.Nonetheless, it was found that the strategy of delivering the protein tothe cells using this click chemistry strategy is effective.

To further ascertain that this delivery is indeed aided by theconjugation to the polymer, also compared was the cellular apoptosiswith the control, where the cells were incubated with the modifiedprotein by itself of similar concentration. This formulation exhibitedno discernible cellular apoptosis, confirming that the polymer is aidingthe cellular uptake. Note also that the polymer-protein conjugate withthe boronic acid moiety without any other degradable linker wouldrelease the protein inside the cells, only in the presence of oxidativestress induced by PMA (FIG. 4 ). In the case of the current system withan engineered disulfide bond, an intrinsic intracellular stimulus in GSHwas utilized. Therefore, the protein is activated in this case withoutthe need for PMA-based activation of the cells. This feature furthersupports the disclosed self-assembly and stimuli-induced disassemblymechanisms outlined here. Overall, the molecular design principle andthus the generality of the design strategy are successfully demonstratedin this system.

In order to increase the delivery efficiency, additional components wereincorporated into the polymer chain through copolymerization ofdifferent functional monomers. Guanidium functionality was incorporatedinto the polymers for efficient delivery. P3 was synthesized though aRAFT polymerization of three monomers of trt-protected SaH (10%), PEG-MA(30%) and Boc protected guanidium monomer (60%) with subsequent acidcleavage of Trt protection group. GFP was chosen as the model protein totest the intracellular delivery efficiency, because of its intrinsicfluorescence. The surface lysines of GFP were modified by boronic acidfor the complexation with polymer P3 via the samesalicylhydroxamate-boronic acid reversible click reaction. The resultantnanoassembly was incubated with HeLa cells for intracellular deliverystudy and the efficiency was evaluated with confocal microscopy. Theresults showed that the cellular uptake was indeed significantlyenhanced. For control, GFP lacking the boronic acid surface modificationand thus cannot undergo the covalent click complexation with thepolymer, was also tested for intracellular delivery in the presence ofpolymer. Intracellular delivery of the protein was found to benegligible, if any. These results indicate that the present strategyenhances efficiency of intracellular protein delivery. However,positively charged polymers usually reveal significant toxicity becauseof its electrostatic interaction with the cellular membranes. Thecytotoxicity of the polymer was checked through MTT assay. Indeed, thismaterial exhibited a very high toxicity.

Other functional groups were explored that could enhance the cellularuptake without significant toxicity. The new polymer P4 was designed byincorporation of the fluorine-component in the polymer chain, whileretaining the salicylhydroxamate for efficient protein conjugation withPEG component for water solubility and compatibility. Polymer P4 wassynthesized though a RAFT polymerization of the three components oftrt-protected SaH (10%), PEG-MA (30%) and tri-fluoromonomer (60%) withsubsequent acid cleavage of Trt protection group. The cell uptake of theGFP protein was accessed to evaluate the delivery efficiency by thispolymer. However, the incorporation of the fluorine-component itselfcould not enhance the uptake.

It is noted that the literature works were focused on thepost-modification of positive charged materials (e.g., PAMAM dendrimersand PEI) with the fluoro-component for enhanced delivery, while thepolymer was a neutral one without the charge moiety. Therefore, bothguanidium groups and fluorocarbon moieties were incorporated onto thepolymer chain. At first, a polymer P5 was designed with 40% fluorinemoiety, 20% guanidium functionality, while retaining 30% PEG and 10% ofSaH for study. The intracellular delivery efficiency was also tested byconfocal microscopy and flow cytometry. Incorporation of both guanidiumand fluorine moieties do enhance the intracellular delivery of theproteins, compared to polymers P1 and P4. The cytotoxicity of thepolymer was also tested. Interestingly, this polymer does not exhibitany discernible cytotoxicity even at the concentrations as high as 1mg/mL. However, the delivery efficiency was much lower than P3 with veryhigh percentage of guanidium moiety inside the chain.

Considering these results, a series of polymers were explored withsystematically tuned structural features in order to achieve enhanceddelivery efficiency. Factors that are considered in thisstructure-property relationship study include hydrophobicity of thepolymers (P5-P7), charge density of the polymers (P5, P8, P9) andfluorine chain length (P10-P12). Some of the polymers (P8-P12) exhibitvery high delivery efficiency, where 2-3 orders of cellular uptakeenhancement was observed compared to P1.

The structures of various exemplified polymers are provided in FIG. 56 .

Polymerizations were carried out underreversible-addition—fragmentation-chain-transfer (RAFT) reactionconditions. Deprotections of Boc-and Trityl-protected copolymer wereperformed in TFA solution. The composition of each moiety was measuredby proton nuclear magnetic resonance (¹H NMR), which was consistent withthe feed ratio of the monomers.

Polymer structures of P3′-P12′ are shown in FIG. 57 .

Scheme 3 shows exemplary synthetic approach to certain polymers P3′-P9′discussed herein.

A reactive self-assembly process, based on the ultrafast click reactionbetween salicylhydroxamic acid and boronic acid moieties, can beutilized for generating functional polymer-protein conjugatenanoassemblies. Polymer-protein nanoconjugates can be quantitativelygenerated using a simple ‘mix and go’ approach, where boronicacid-modified proteins react with salicylhydroxamic acid side chainfunctionalities of a polymer. The versatility of this reactiveself-assembly approach has been demonstrated through variations inprotein structures, their responsive behavior to a variety of stimuli,and utility in intracellular transport of proteins. Also shown was thatproteins of different sizes (from ˜14 kDa to ˜400 kDa) can be utilizedin the self-assembly based encapsulation. Both cationic and anionicproteins can be utilized to form these nanoassemblies with practicallyindistinguishable efficiencies. In addition, the conjugation can befully reversed, to cause programmed disassembly of the conjugate andthus releasing the proteins from the polymer. The linkers have beenengineered such that this process results in traceless release ofproteins, where there are no remnants of functional group modificationson protein surfaces. In addition to exploiting the inherentsusceptibility of boronic acids to oxidants to cause thisstimulus-triggered disassembly, self-immolative linkers were introducedto expand the sensitivity of the nanoassemblies to other stimuli, suchas pH and reducing conditions. Overall, this work shows that reactionbetween polymers and proteins can be quite efficient, when the reactivefunctional groups are judiciously chosen to overcome the sparsefunctional group densities and low reaction concentration requirements.The synthetic approach and the subsequent self-assembly process,involving polymers and proteins, have implications in a variety ofresearch areas including triggerable catalysis and biologics delivery.Also demonstrated was the direct implications of the self-assemblystrategy with intracellular delivery of proteins into mammalian cells.

Experimental

Materials and instruments. Unless mentioned, all chemicals and proteinswere used as received from Sigma-Aldrich. ¹H-NMR and ¹³C-NMR spectrawere recorded on a 400 MHz Bruker NMR spectrometer. Molecular weight ofthe polymers was measured by gel permeation chromatography (GPC, Waters)using a PMMA standard with a refractive index detector and THF as eluentwith a flow rate of 1 mL/min. Dynamic light scattering (DLS)measurements were performed using a Malvern Nanozetasizer. UV-visibleabsorption spectra were recorded on a Varian (model EL 01125047)spectrophotometer. The fluorescence spectra were obtained using a JASCOFP-6500 spectrofluorimeter. Fluorescent images were recorded on Nikonwith Yokogawa spinning disk (SD) and Nikon A1 spectral detector confocalwith FLIM module.

Protein modification by ROS sensitive linker. 3 mg of RNase A wasdissolved in 0.5 mL of 0.1 M NaHCO₃ buffer solution (pH=8.5). To theabove solution was added 150 μL of DMSO solution containing 4.8 mgcompound 1. The reaction mixture was then stirred at ambient temperaturefor an additional 10 h, followed by filtration with a 220 μm filter andan ultrafiltration purification using Amicon® Ultra Centrifugal Filters(MWCO=3,000). The final protein was dissolved in 300 μL of DI water (10mg/mL) and stored at 4° C. The modification of the boronic acid linkerwas quantified by MADLI-MS. Other proteins such as GFP, BSA, and β-galwere prepared using the similar method and also stored at 4° C. with theconcentration of 10 mg/mL. The boronic acid modified proteins with ROSresponsiveness (RNase A, GFP, BSA, β-gal) are denoted as RNaseA-BA,GFP-BA, BSA-BA, β-gal-BA.

Protein modification by redox and pH sensitive linker. The method formodification of proteins with redox and pH sensitive linkers is similarto that for ROS sensitive linker by using compound 6 and 7 for reaction.For instance, 3 mg of RNase A was dissolved in 0.5 mL of 0.1 M NaHCO₃buffer solution (pH=8.5). To the above solution was added a 150 μL ofDMSO solution containing 6 mg of compound 6 or 4 mg of compound 7. Thereaction mixture was then stirred at room temperature for additional 10h, followed by filtration on 220 μm filter and ultrafiltrationpurification using Amicon® Ultra Centrifugal Filters (MWCO=3,000). Thefinal protein was dissolved in 300 μIL DI water (10 mg/mL) and stored at4° C. The modification was quantified by MADLI-MS. The modified RNase Awith redox sensitive linker is referred to RNase A-SS-BA. The pHsensitive linker modified RNase A is referred to RNase A-BA_pH.

Labeling of proteins with Rhodamine B. To perform the cellular uptakestudies, fluorescently-labelled proteins (RNase A, BSA, RNase A-BA andBSA-BA) were prepared by reaction with rhodamine B (RB) isothiocyanate.In a typical labelling procedure, proteins (3 mg) were dissolvedseparately in 2 mL of 0.1 M NaHCO₃ buffer (pH 8.5) under stirring. RBisothiocyanate (5 eq. of each protein, 10 mg/mL in DMSO) was addeddropwise to the protein solution and stirred at ambient temperature for2 h in dark. The RB-labelled-proteins were purified by extensiveultrafiltration purification using Amicon® Ultra Centrifugal Filters(MWCO=3,000) to remove excess RB.

MALDI-MS for quantification of the modification. The surface functionalboronic acid modification of the proteins was quantified by MALDI-MS.MALDI-MS analyses were performed with Bruker Autoflex III time-of-flightmass spectrometer. All mass spectra were acquired in reflectron modewith an average of 500 laser shots at ˜60% optimized power.

Preparation of protein-polymer nanoassemblies. Protein-polymernanoassemblies were prepared by mixture of the boronic acid modifiedprotein and polymer with different mass ratios at ambient temperaturefor 12 h.

Fluorescence polarization measurement. Fluorescence polarization wasused to monitor the complexation kinetics of the polymer and boronicacid modified protein. GFP was chosen to study the fluorescentpolarization because of its intrinsically strong fluorescence.Fluorescence polarization was monitored using a SpectraMax M5 platereader with a fixed excitation wavelength set to 480 nm and an emissionwavelength set to 520 nm. Samples (GFP, GFP-BA, GFP+polymer, andGFP-BA+polymer) were incubated in 96-well plate and the fluorescencepolarization was measured immediately after placing all the componentstogether with an interval of 30 s. The process lasted for 2 h at ambienttemperature. The ratio between the protein and polymer was 1:10.

Fluorescence titration by Alizarin Red S (ARS) assay. To further monitorthe protein-polymer complexation, 0.0025% w/v ARS solution was incubatedwith 0.25 mg/mL of RNase A-BA for 2 h. This solution was furthertitrated with different amounts of polymer. In all processes, theconcentrations of the RNase A-BA and ARS were kept constant. After thepolymer was incubated for another 15 min, ARS fluorescence was monitoredat 600 nm with excitation at 490 nm.

ROS-responsive study of RNaseA-BA and related nanoassemblies. RNase A-BAand RNase A-BA@polymer (protein and polymer ratio was 1:10) wereincubated with 10 mM of H₂O₂ at ambient temperature for 12 h, followedby ultrafiltration purification using Amicon® ultracentrifugal filters(MWCO=3,000). The proteins were then subjected to ESI-MScharacterization or enzyme activity assay and compared to modifiedproteins without H₂O₂ treatment or native RNase A.

SDS-PAGE gel for protein-polymer complexation and release studies. 20 μLof different samples were mixed with 5 μL of loading buffer and 20 μL ofeach sample was loaded on acrylamide gel. The protein-polymer mixturewas prepared by incubation of the polymer and boronic acid modifiedprotein for 12 h.

For complexation kinetics, the polymer and the protein were mixed in acentrifuge tube for different times. At the desired time, the complexwas mixed with loading buffer and was characterized with a run in thegel immediately.

For ROS responsive release experiment, identical protein-polymerconjugate samples (after incubation for 12 h) were treated withdifferent amounts of H₂O₂ (1 mM or 10 mM) and incubated at roomtemperature for different time intervals, before subjecting toacrylamide gel electrophoresis. To calculate the amount of releasedprotein from each sample, standard curves were generated from the knownconcentrations of pure protein samples loaded into the gel lanes. Thegel image analysis and quantification were performed with Bio-Rad ImageLab™ software.

For redox and pH responsive release experiment, the protein-polymercomplexes were treated with DTT (or GSH, 10 mM) and pH=5.0 for 12 h,before subjecting to acrylamide gel electrophoresis.

Activity assay. For RNase A: RNaseAlert® activity kit (Thermo-FisherScientific) was used to check the activity of RNase A based samples.RNase A cleaves the oligonucleotide substrate consisting a fluorophoreand a quencher present at two ends, thus releasing the fluorophore whichcan be detected and quantified with a fluorimeter. For a typicalkinetics experiment, 5 μL of RNaseAlert® substrate and 10 μL of assaybuffer (10×, provided by the assay kit) were placed in a black 96-wellplate. To the above assay substrate, 85 μL of RNase A containing samples(2 ng/mL of final proteins) were added. The fluorescence intensity at520 nm (Excitation 490 nm) was monitored with SpectraMax® M5spectrophotometer over 30 min time.

For β-gal: Beta-galactosidase (β-gal) activity assay kit (BioVision) wasused to measure the activity of β-gal based samples.\, where thefluorescence (Ex/Em=480/520 nm) was monitored in kinetic mode for 30min. For a typical kinetics experiment, 2 μL of RNaseAlert® Substrateand 97 μL of assay buffer (provided by the assay kit) were placed in ablack 96-well plate. To the above assay substrate, 1 μL of β-galcontaining samples (0.5 nM of final proteins for β-Gal, β-Gal-BA,β-Gal-BA@polymer) were added.

Circular dichroism (CD) spectra. CD spectra of the protein complex,released protein and native protein samples were recorded on JASCOJ-1500 spectrophotometer. The protein complex was prepared through thetypical complexation method by mixture of the boronic acid modifiedprotein and polymer with the ratio of 1:10 for 12 h. The releasedprotein sample was prepared by incubation of the protein-polymer samplein 10 mM H₂O₂ for 24 h. It was further purified by ultracentrifuge withAmicon Ultra Centrifugal Filters MWCO 3k for 5 times. For recording thespectra, 200 μL of protein solution was injected into a quartz cuvetteof 1-mm path length, equilibrated at 25° C. for 10 min and scanned from190 to 250 nm (scan rate: 20 nm/min, interval: 0.2 nm, average of threespectra).

Cell culture. Different cell lines (including human cervical carcinomaHeLa cells, MDA-MB-231 cells and MCF-7 cells) were cultured in T75 cellculture flask containing Dulbecco's Modified Eagle Medium/NutrientMixture F-12 (DMEM/F12) in a humidified S26 incubator with 5% CO₂ at 37°C. Culture media was supplemented with 10% fetal bovine serum (FBS), 1%L-glutamine and 1% antibiotic-antimycotic (100 units/mL of penicillin,100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B).

Cellular uptake studies and endosomal escape experiment. Cellinternalization studies were performed with HeLa cells, seeded at150,000 cells/mL in glass-bottomed petri-dishes and cultured for 24 h at37° C. in a 5% CO₂ incubator. Prior to delivery, cells were washed threetimes with PBS buffer and incubated with 1 mL media containingpolymer-rhodamine B-protein conjugate or rhodamine B-protein conjugate(protein concentration 30 μg/mL) at 37° C. for 6 h. After that, cellnucleus was stained with Hoechst 33342 (8 μM) and finally the media wasreplaced with fresh stock and incubated for another 1 h beforesubjecting to CLSM analysis. In addition, to studying the endosomalescape of the labelled proteins, HeLa cells were incubated with labellednanoassemblies for 4 and 24 h. After that, it was stained withLysoTracker® Green to label endosomes/lysosomes and studied theco-localization of red and green fluorescence channels. Live cellimaging was performed using Nikon Spectral A1 confocal microscope.

Cell viability by MTT assay. Different cells (including HeLa cells,MDA-MB-231, MCF-7) were seeded into 96-well tissue culture plates at adensity of 10,000 cells/well/100 μL sample and incubated at 37° C. After24 h, culture media was replaced and cells were treated with differentconcentrations of protein-polymer complex and control protein samples(0.1 mg/mL to 2 mg/mL protein-polymer complex; naked protein andmodified protein concentrations were matched with the protein-polymercomplex) in 100 μL media (10 μL protein containing solution withdifferent concentrations+90 μL medium). At the desired time interval,the medium was removed and the cells were cultured by 100 μL 10% MTT (5mg/mL) in medium solution for another 4 h. Then, the solution wasdiscarded and the remaining crystal was dissolved by 100 μL DMSO. Thesolution was subjected to absorbance measurement with SpectraMax® M5 at590 nm. Cell death was measured by the MTT assay in triplicate.

Cell viability after PMA treatment for ROS sensitive nanoassembly. HeLacells were seeded in a 96-well plate for 24 h, before the deliveryexperiment was performed at a density of 10,000 cells per well (100 μL).On the day of the experiment, cells were pre-treated with RNaseA-BA@polymer nanoparticles with varied concentrations for 24 h. Afternanoparticle removal, cells were then treated with 200 nM PMA in DMEM(or DMEM only as a control) for 1 h. After removing PMA and washing thecells with DMEM, cells were maintained for another 24 h with freshculture medium before cell viability measurement using the MTT assay.The toxicity of PMA was excluded by treating cells with 200 nM PMA inthe absence of RNase A complex.

Additional Experimental Synthesis of the ROS Responsive Linker

Molecule 1 was synthesized according the reported method. (Jourden, etal. Angew. Chem. Int. Ed. 2010, 49, 6795-6797.)

Molecule 1: ¹H NMR (400 MHz, CDCl₃) (δ ppm): 8.25 (d, J=9.1 Hz, 2H),7.85 (d, J=7.9 Hz, 2H), 7.43 (d, J=7.8 Hz, 2H), 7.36 (d, J=9.1 Hz, 2H),5.30 (s, 2H), 1.35 (s, 12H).

Synthesis of the Salicylhydromate Functional Monomer

Molecule 3a and 3b were synthesized according to the literature, and the¹H NMR spectra were consistent with the reported results. (Ng, et al.Angew. Chem. Int. Ed. 2014, 53, 324-328.)

Molecule 3a: ¹H NMR (400 MHz, CDCl₃) (δ ppm): 7.65-7.55 (m, 4H),7.56-7.52 (m, 6H), 7.31-7.27 (m, 9H).

Molecule 3b: ¹H NMR (400 MHz, CDCl₃) (δ ppm): 7.48-7.40 (m, 6H),7.38-7.24 (m, 9H), 4.94 (s, 2H).

Molecule 3c was also synthesized according to the reported method.(Wisastra, et al. Bioorganic & Medicinal Chemistry, 2012, 20,5027-5032.)

Molecule 3c: ¹H EINMR (400 MHz, MeOD) (δ ppm): 7.73 (d, J=8.4 Hz, 1H),6.58 (d, J=8.1 Hz, 1H), 6.35 (s, 1H), 1.69 (s, 6H).

Synthesis of Molecule 3d

To a solution of 2-bromoethanol (8.0 g, 4.54 mL, 64.0 mmol) in 50 mL ofdry dichloromethane was added 8.42 g (11.6 mL, 83.2 mmol) oftriethylamine and the mixture was cooled in an ice-bath. To this coldmixture, a solution of methacryloyl chloride (8.03 g, 7.51 mL, 76.8mmol) in 10 mL dichloromethane was added drop-wise with continuousstirring. After the addition was over the reaction mixture was stirredat room temperature for overnight. The stirring was stopped and thereaction mixture was washed with saturated NaHCO₃ solution (twice),distilled water and finally with brine. The organic layer was collected,dried over anhydrous Na₂SO₄ and concentrated to get the crude product.It was further purified by combiflash using silica gel as stationaryphase and mixture of ethyl acetate/hexane as eluent. (7.2 g, Yield: 60%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 6.17 (p, J=1.4 Hz, 1H), 5.62 (p, J=1.5Hz, 1H), 4.45 (t, J=6.1 Hz, 2H), 3.55 (t, J=6.1 Hz, 2H), 1.98-1.94 (m,3H).

¹³C NMR (100 MHz, CDCl₃) (δ ppm): 169.97, 135.92, 126.47, 64.08, 28.86,18.36.

Synthesis of Molecule 3e

Compound 3c (2.0 g, 10.3 mmol, 1 equiv), compound 3d (2.0 g, 10.3 mmol,1 equiv), K₂CO₃ (7.12 g, 51.5 mmol, 5 equiv) and 18-Crown-6 (0.27 g, 1.0mmol, 0.1 equiv) were mixed together in acetone (60 mL) and refluxed for20 h. The reaction mixture was concentrated in vacuo and residue wasdissolved in water and the product was extracted 3 times with ethylacetate. The organic layer was collected, dried over anhydrous Na₂SO₄and concentrated to get the crude product. It was further purified bycombiflash using silica gel as stationary phase with ethylacetate/hexane as eluent. (2.46 g, Yield: 78%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 7.88 (d, J=8.7 Hz, 1H), 6.67 (dd,J=8.8, 2.4 Hz, 1H), 6.44 (d, J=2.4 Hz, 1H), 6.14 (dd, J=1.5, 1.0 Hz,1H), 5.60 (p, J=1.5 Hz, 1H), 4.51 (dd, J=5.4, 4.2 Hz, 2H), 4.26 (dd,J=5.3, 4.2 Hz, 2H), 1.95 (dd, J=1.5, 1.0 Hz, 3H), 1.72 (s, 6H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 167.36, 166.32, 160.59, 158.80,137.14, 131. 126.09, 111.60, 107.50, 106.98, 102.61, 67.53, 63.56,25.84, 18.35.

Synthesis of Molecule 3

1.0 g compound 3e was dissolved in 5 mL TFA/H₂O (9:1) mix solvent. Themixture was stirred overnight at room temperature. Then, TFA was removedand 5 mL ice cold water was added. The white precipitate was formed andfiltrated, which was further washed with 5 mL ice old water twice. Thesolid product was dried by lyophilizer. (0.84 g, Yield: 96%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 10.63 (s, 1H), 7.83 (d, J=8.8 Hz, 1H),6.67-6.36 (m, 2H), 6.15 (s, 1H), 5.61 (dd, J=3.1, 1.5 Hz, 1H), 4.51 (dd,J=6.0, 3.5 Hz, 2H), 4.39-4.16 (m, 2H), 1.96 (m, 3H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 172.45, 167.49, 165.84, 165.27,137.19, 132.73, 126.03, 108.38, 106.29, 102.22, 67.19, 63.63, 18.35.

Synthesis of Molecule 4

Molecule 4 was prepared through a two-step reaction as shown in Scheme4. 0.5 g compound 3 was dispersed in 20 mL dry DCM under ice bath. Then,0.5 mL oxalyl dichloride was added under stirring. Catalytic amount ofDMF (3 drops) was added into the above mixture for reaction. Thereaction was performed at room temperature for 3 h. The reaction mixturewas concentrated in vacuo to remove the solvent and excess oxalylchloride. The residue was re-dissolved in 10 mL dry DCM. 0.9 g ofcompound 3b was added into the above solution under ice bath. 1.5 mL TEAwas added for reaction. The mixture was allowed to warm up to roomtemperature and reacted for another 1 week. The organic solvent wasremoved and purified by combiflash using silica gel as stationary phasewith ethyl acetate/ hexane as eluent. (0.31 g, Yield: 31%)

¹H NMR (400 MHz, Acetone-d₆) (δ ppm): 11.75 (s, 1H), 10.10 (s, 1H),7.55-7.20 (m, 16H), 6.46-6.32 (m, 2H), 6.05 (dd, J=1.7, 1.0 Hz, 1H),5.65-5.48 (m, 1H), 4.55-4.36 (m, 2H), 4.30-4.24 (m, 2H), 1.91-1.88 (m,3H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 167.37, 164.20, 143.43, 137.19,130.24, 129.69, 128.49 (m), 125.99, 107.45, 102.61, 94.01, 66.98, 63.63,18.33.

Synthesis of the Trityl Protected Polymer (P1′):

A mixture of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (6.2 mg,0.022 mmol), monomer 4 (46.5 mg, 0.089 mmol), monomer 5 (polyethyleneglycol monomethyl ether methacrylate, average Mw: 500, 400 mg, 0.8 mmol)and AIBN (1.46 mg, 0.006 mmol) were dissolved in 0.8 mL THF and degassedwith three freeze-pump-thaw cycles. Then the reaction was transferred toa pre-heated oil bath (65° C.) and stirred for 36 hours under argonatmosphere. The resultant polymer was precipitated in cold diethyletherand washed with the cold ether for several times to remove unreactedmonomers. ¹H NMR spectrum of the resulting polymer is shown in FIG. 19 .Based on the NMR spectrum, the ratio between 4 and 5 was calculatedbased on the integration of peaks at 7.70-7.20 ppm (compound 4, 16 H)and 3.31 ppm (monomer 5, PEG methoxy group, 3H). The monomer ratio inpolymer was calculated to be 1:9, which was the same as the feed ratio.The molecular weight of the polymer was determined by GPC and theresults are shown in FIG. 21 . (Mw=18.8 k, PDI=1.12)

Deprotection of the Polymer (P1):

The polymer was further deprotected under TFA to remove the Trtprotection group. 100 mg of the above synthesized polymer P1′ wasdissolved in 1 mL DCM. Then, 0.5 mL TFA and 0.05 mL triisopropylsilane(TIPS) was added under stirring at room temperature overnight. Then, thedeprotected polymer was precipitated in cold diethylether and theproduct was collected by centrifuge. Further, the polymer was washedwith cold ether for several times and dried under vacuum. Thedisappearance of the aromatic ring peak in the ¹H NMR spectrum at7.70-7.20 ppm for Trt group demonstrated the successful deprotection(FIG. 20 ). The molecular weight of the polymer was determined by GPCand the results were shown in FIG. 21 . (Mw=17.4 k, PDI=1.15)

Synthesis of the Rhodamine B Dye Labelled Polymer (P2):

A mixture of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (6.2 mg,0.022 mmol), monomer 4 (46.5 mg, 0.089 mmol), monomer 5 (polyethyleneglycol monomethyl ether methacrylate, average Mw: 500, 400 mg, 0.8mmol), Rhodamine B monomer (6.1 mg, 0.0089 mmol) and AIBN (1.46 mg,0.006 mmol) was dissolved in 0.8 mL THF and degassed with threefreeze-pump-thaw cycles. Then the reaction was transferred to apre-heated oil bath (65° C.) and stirred for 36 hours under argon. Theresultant polymer was precipitated in cold diethylether and washed withthe cold ether for several times to remove unreacted monomers. The ¹HNMR of P2′ was almost the same as P1 as shown in FIG. 19 . The ratiobetween 4 and 5 was in polymer was also calculated to be 1:9, which wasthe same as the feed ratio. The molecular weight of the polymer wasdetermined by GPC and the results were shown in FIG. 21 , which was verysimilar to P1. (Mw=17.0 k, PDI=1.12)

Deprotection of the Rhodamine B Dye Labelled Polymer (P2):

Further, the rhodamine B labelled polymer was also deprotected under TFAto remove the Trt protection group. 100 mg of the above synthesizedpolymer P2′ was dissolved in 1 mL DCM. Then, 0.5 mL TFA and 0.05 mLtriisopropylsilane (TIPS) was added under stirring at room temperatureovernight. Then, the polymer was precipitated in cold diethylether andthe product was collected by centrifuge. Further, the polymer was washedwith cold ether for several times and dried under vacuum. The molecularweight of the polymer was determined by GPC and the results were shownin FIG. 21 . (Mw=18.3 k, PDI=1.16)

Synthesis of the pH Responsive Linker:

Synthesis of Molecule 6

Molecule 6 was prepared through a two-step reaction as shown in Scheme8. 0.334 g cis-aconitic anhydride (2.1 mmol) was dissolved in 10 mL dryDCM under ice bath. Then, 0.4 mL oxalyl dichloride was added understirring. Catalytic amount of DMF (3 drops) was added into the abovemixture for reaction. The reaction was performed at room temperature for3 h. The reaction mixture was concentrated in vacuo to remove thesolvent and excess oxalyl chloride. The residue was redissolved in 10 mLdry DCM. 0.5 g of 4-(Hydroxymethyl)phenylboronic acid pinacol ester (2.1mmol) was added into the above solution under ice bath. 1 mL TEA wasadded for reaction. The mixture was allowed to warm up to roomtemperature and reacted for another 24 h. The organic solvent wasremoved and purified by combiflash using silica gel as stationary phasewith ethyl acetate/hexane as eluent. (0.16 g, Yield: 20%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 7.82 (d, J=8.0 Hz, 2H), 7.35 (d, J=8.0Hz, 2H), 7.00 (t, J=1.7 Hz, 1H), 5.21 (s, 2H), 3.63 (d, J=1.7 Hz, 2H),1.35 (s, 12H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 168.29, 166.59, 165.05, 146.40,139.82, 135.65, 132.50, 132.46, 128.17, 84.62, 67.54, 31.35, 25.18.

Synthesis of the Redox Responsive Linker:

Synthesis of Molecule 7a

3.32 g 4-boronobenzoic acid (20.1 mmol), 2.37 g pinacol (20.1 mmol) and10 g 5 Å molecular sieve were dispersed in 40 mL dry DCM. The reactionmixture was stirred at room temperature for overnight. Then, thereaction solution was filtered to remove molecular sieves and all solidimpurities. The solid was rinsed by DCM 3 times. The combined solutionwas concentrated to get the product. (Yield: quantitative)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 8.09 (d, J=7.7 Hz, 2H), 7.90 (d, J=7.6Hz, 2H), 1.36 (s, 12H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 167.67, 135.42, 133.77, 129.57,84.94, 25.18.

Synthesis of Molecule 7b

1 g compound 7a (4.0 mmol), 1.244 g 2-Hydroxyethyl disulfide (8.0 mmol)and 0.049 g DMAP (0.4 mmol) were dissolved in 100 mL DCM under ice bath.1.0 g EDC·HCl 5.2 mmol) was added. The reaction was warmed up to roomtemperature and stirred for 24 h. Then, the reaction mixture was washedby 1 M HCl, saturated NaHCO₃ solution (twice) and brine. The organiclayer was collected, dried over anhydrous Na₂SO₄ and concentrated to getthe crude product. It was further purified by combiflash using silicagel as stationary phase and mixture of ethyl acetate/hexane as eluent.(0.99 g, Yield: 64%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 8.00 (d, J=8.1 Hz, 2H), 7.85 (d, J=8.0Hz, 2H), 4.57 (t, J=6.7 Hz, 2H), 3.86 (t, J=5.9 Hz, 2H), 3.04 (t, J=6.7Hz, 2H), 2.87 (t, J=5.9 Hz, 2H), 1.33 (s, 12H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 166.55, 135.47, 133.28, 129.37,84.99, 63.70, 60.97, 42.50, 37.79, 25.18.

Synthesis of Molecule 7

0.5 g compound 7b (1.3 mmol) and 0.263 g TEA (0.363 mL, 2.6 mmol) weredissolved in 5 mL dry DCM under ice bath. 0.278 g 4-nitrochloroformate(1.4 mmol) was added for reaction. The reaction was stirred under roomtemperature for overnight. Then, the reaction mixture was washed withsaturated NaHCO₃ solution (twice) and brine. The organic layer wascollected, dried over anhydrous Na₂SO₄ and concentrated to get the crudeproduct. It was further purified by combiflash using silica gel asstationary phase and mixture of ethyl acetate/hexane as eluent. (0.404g, Yield: 57%)

¹H NMR (400 MHz, CDCl₃) (δ ppm): 8.28-8.20 (m, 2H), 8.01 (d, J=8.2 Hz,2H), 7.86 (d, J=8.1 Hz, 2H), 7.39-7.33 (m, 2H), 4.60 (t, J=6.6 Hz, 2H),4.54 (t, J=6.5 Hz, 2H), 3.10 (t, J=6.6 Hz, 2H), 3.05 (t, J=6.5 Hz, 2H),1.35 (s, 12H).

¹³C NMR (100 MHz, Acetone-d₆) (δ ppm): 166.56, 156.62, 153.17, 146.46,135.48, 133.21, 129.39, 126.08, 123.14, 85.01, 67.66, 54.96, 37.79,37.61, 25.18.

Synthesis of P6′

A mixture of 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (4.18mg, 0.015 mmol), AIBN (0.99 mg, 0.006 mmol), monomer M1 (polyethyleneglycol monomethyl ether methacrylate, average MW: 500, 375 mg, 0.75mmol), monomer M2 (78.54 mg, 0.15 mmol), Boc-protected guanidium monomerM3 (111.42 mg, 0.3 mmol) and 2,2,2-Trifluoroethyl methacrylate M4 (50.43mg, 0.3 mmol) were dissolved in 1.5 mL THF and degassed with threefreeze-pump-thaw cycles. Then the reaction was transferred to apre-heated oil bath (65° C.) and stirred for 20 hours under argon. Theresultant polymer P6′ was precipitated in cold hexanes and washed withhexanes for several times to remove unreacted monomers.

Synthesis of P3′-P9′

The procedures of P3′-P9′ were similar to the one used for the synthesisof P6′. The dosage and ratios of monomers were according to Table 2, andother feed reagents and the synthetic routes were same with P6′.

TABLE 2 M1/ M1/ M2/ M2/ M3/ M3/ M4/ M4/ mg mmol mg mmol mg mmol mg mmolP3′ 225 0.45 78.5 0.15 334.2 0.9 0 0 P4′ 225 0.45 78.5 0.15 0 0 151.20.9 P5′ 225 0.45 78.5 0.15 111.4 0.3 100.8 0.6 P6′ 375 0.75 78.5 0.15111.4 0.3 50.4 0.3 P7′ 150 0.3 78.5 0.15 111.4 0.3 126 0.75 P8′ 150 0.378.5 0.15 167.1 0.45 100.8 0.6 P9′ 75 0.15 78.5 0.15 222.8 0.6 100.8 0.6

Synthesis of P10′-P12′

The procedure used for synthesis of P10′, P11′, P12′ was similar to thatfor P6′. M1 (150 mg, 0.3 mmol), M2 (78.5 mg, 0.15 mmol) and M3 (167.1mg, 0.45 mmol) were mixed with M5 (2,2,3,3,3-Pentafluoropropylmethacrylate, 130.7 mg, 0.6 mmol), M6 (2,2,3,3,4,4,4-Heptafluorobutylmethacrylate, 168.8 mg, 0.6 mmol) and M7(2,2,3,3,4,4,5,5-Octafluoropentyl methacrylate, 190.0 mg, 0.6 mmol), forsynthesis of P10′, P11′ and P12′ respectively. Other reagents (AIBN andRAFT reagent) and synthetic procedures were the same as P6′ describedabove.

Deprotection of the Polymers to Make P3-P12

The polymers were further deprotected under TFA to remove the Trt andBoc protection groups. 200 mg of the above synthesized polymer(P3′-P12′) was dissolved in 2 mL DCM. Then, 2 mL TFA and 0.15 mLtriisopropylsilane (TIPS) was added under stirring at room temperature.The reaction was stirred for overnight. Then, the deprotected polymerwas dried by air flow. The residue was dissolved in THF. The resultantsolution was precipitated in hexanes and the product was collected bycentrifuge. Further, the obtained polymer was washed with hexanes forseveral times and dried under vacuum.

Protein Modification by ROS Sensitive Linker

3 mg RNase A was dissolved in 0.5 mL 0.1 M NaHCO₃ buffer solution(pH=8.5). To above solution was added 150 μL DMSO solution containing4.8 mg compound 8. The reaction mixture was then stirred at roomtemperature for additional 10 h, followed by filtration with a 220 μmfilter and ultrafiltration purification using Amicon® Ultra CentrifugalFilters (MWCO=3,000). The final protein was dissolved in 300 μL DI water(10 mg/mL) and stored at 4° C. The modification of the boronic acidlinker was quantified by MADLI-MS.

Other proteins such as GFP, BSA, β-gal were prepared using the similarmethod and also stored at 4° C. with the concentration of 10 mg/mL. Theboronic acid modified proteins with ROS responsiveness (RNase A, GFP,BSA, β-gal) are denoted as RNase A-BA, GFP-BA, BSA-BA, β-gal-BA.

Labeling of Proteins With Rhodamine B

To perform the cellular uptake studies, fluorescently-labelled proteins(RNase A, BSA, RNase A-BA and BSA-BA) were prepared by reaction withrhodamine B isothiocyanate (RB). In a typical labelling procedure,proteins (3 mg) were dissolved separately in 2 mL of 0.1 M NaHCO₃ buffer(pH 8.5) under stirring. RB (5 eq. of each protein, 10 mg/mL in DMSO)was added dropwise to each protein solution and stirred at roomtemperature for 2 h under the protection from light. TheRB-labelled-proteins were purified by extensive ultrafiltrationpurification using Amicon® Ultra Centrifugal Filters (MWCO=3,000) toremove excess RB.

Redox and pH Sensitive Linker Modification

The method for modification of protein with redox sensitive and pHsensitive linker is the same as that for ROS sensitive linker by usingcompound 11 and 12 for reaction. 3 mg RNase A was dissolved in 0.5 mL of0.1 M NaHCO₃ buffer solution (pH=8.5). To above solution was added to a150 μL DMSO solution containing 6 mg compound 11 or 4 mg compound 12.The reaction mixture was then stirred at room temperature for additional10 h, followed by filtration on 220 μm filter and ultrafiltrationpurification using Amicon® Ultra Centrifugal Filters (MWCO=3,000). Thefinal protein was dissolved in 300 μL DI water (10 mg/mL) and stored at4° C. The modification was quantified by MADLI-MS. The modified RNase Awith redox sensitive linker is referred to as RNase A-SS-BA. The pHsensitive linker modified RNase A is referred to as RNase A-BA_pH.

MALDI-MS For Quantification of the Modification

The surface functional boronic acid modification of the proteins wasquantified by MALDI-MS. MALDI-MS analyses were performed with BrukerAutoflex III time-of-flight mass spectrometer. All mass spectra wereacquired in the reflectron mode with an average of 500 laser shots at˜60% optimized power.

Protein-Polymer Complex

Protein polymer complexes were prepared by mixture of the boronic acidmodified protein and polymer with different mass ratios at roomtemperature for 12 h.

Fluorescence Polarization Measurement

Fluorescence polarization was used to monitor the complexation kineticsof the polymer and boronic acid modified protein. GFP was chosen tostudy the fluorescent polarization as its intrinsic strong fluorescence.Fluorescence polarization was monitored using a SpectraMax M5 platereader with a fixed excitation wavelength set to 480 nm and an emissionwavelength set to 520 nm. Samples (GFP, GFP-BA, GFP+polymer, andGFP-BA+polymer) were incubated in 96-well plate and the FP was measuredimmediately after placing all the components together with an intervalof 30 s. The process lasted for 2 h at room temperature. The ratiobetween the protein and polymer was 1:10.

Fluorescence Titration by Alizarin Red S (ARS) Assay:

To further monitor the protein polymer complexation, 0.0025% w/v ARSsolution was incubated with 0.25 mg/mL of RNase A-BA for 2 h. Thissolution was further titrated by different amounts of polymer. In allprocesses, the concentrations of the RNase A-BA and ARS were keptconstant. After the polymer was incubated inside the solution foranother 15 min, ARS emission measurement was performed by fluorometer.Emission was monitored at 600 nm with excitation at 490 nm.

ROS-Responsive Study of Modified RNase A:

RNase A-BA and RNase A-BA@polymer (protein and polymer ratio is 1:10)were incubated with 10 mM H₂O₂ at room temperature for 12 h, followed byultrafiltration purification using Amicon® Ultra Centrifugal Filters(MWCO=3,000). The proteins were then subjected to ESI-MScharacterization or enzyme activity assay and compared to modifiedproteins without H₂O₂ treatment or native RNase A.

SDS-PAGE For Protein-Polymer Complexation and Release Studies:

20 μL of different samples were mixed with 5 μL of loading buffer and 20μL of each sample was loaded on acrylamide gel. The protein-polymermixture was prepared by incubation of the polymer and boronic acidmodified protein for 12 h.

For complexation kinetics, the polymer and the protein were mixed in acentrifuge tube for different times. At the desired time, the complexwas mixed with loading buffer and run the gel immediately.

For ROS responsive release experiment, identical protein-polymerconjugate samples (after incubation for 12 h) were treated withdifferent amounts of H₂O₂ (1 mM or 10 mM) and incubated at roomtemperature for different time intervals before subjecting to acrylamidegel electrophoresis. To calculate the amount of released protein fromeach sample, standard curves were generated from the knownconcentrations of pure protein samples loaded into the gel lanes. Thegel image analysis and quantification were performed with Bio-Rad ImageLab™ software.

For redox and pH responsive release experiment, the protein-polymercomplexes were treated with DTT (or GSH, 10 mM) and pH=5.0 for 12 hbefore subjecting to acrylamide gel electrophoresis.

Activity Assay: For RNase A

RNaseAlert® activity kit (Thermo-Fisher Scientific) was used to checkthe activity of RNase A based samples. RNase A cleaves theoligonucleotide substrate of the assay consisting a fluorophore and aquencher present at two extreme ends, thus releasing the fluorophorewhich can be detected and quantified with a fluorometer. For a typicalkinetics experiment, 5 μL RNaseAlert®Substrate and 10 μL assay buffer(10×, provided by the assay kit) were placed in a black 96-well plate.To the above assay substrate, 85 μL of RNase A containing samples (2ng/mL of final proteins) were added. The fluorescence intensity at 520nm (Excitation 490 nm) was monitored with SpectraMax® M5spectrophotometer over 30 min time.

For/β-Gal

Beta galactosidase (β-Gal) activity assay kit (BioVision) was used tomeasure the activity of β-Gal based samples. Measure fluorescence(Ex/Em=480/520 nm) immediately in kinetic mode for 30 min. For a typicalkinetics experiment, 2 μL RNaseAlert® Substrate and 97 μL assay buffer(provided by the assay kit) were placed in a black 96-well plate. To theabove assay substrate, 1 μL of β-Gal containing samples (0.5 nM of finalproteins for β-Gal, β-Gal-BA, β-Gal-BA@polymer) were added.

Circular Dichroism (CD) Spectra:

CD spectra of the protein complex, released protein and native proteinsamples were recorded on JASCO J-1500 spectrophotometer. The proteincomplex was prepared through the typical complexation method by mixtureof the boronic acid modified protein and polymer with the ratio of 1:10for 12 h. The released protein sample was prepared by incubation of theprotein-polymer sample with 10 mM H₂O₂ for 24 h. It was further purifiedby ultracentrifuge with Amicon Ultra Centrifugal Filters MWCO 3K for 5times. For recording the spectra, 200 μL protein solution was injectedinto a quartz cuvette of 1-mm path length, equilibrated at 25° C. for 10min and scanned from 190 to 250 nm (scan rate: 20 nm/min, interval: 0.2nm, average of three spectra).

Cell Culture:

Different cell lines (including Human cervical carcinoma (HeLa) cells,MDA-MB-231 cells and MCF-7 cells) were cultured in T75 cell cultureflask containing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12(DMEM/F12) in a humidified S26 incubator with 5% CO₂ at 37° C. Culturemedia was supplemented with 10% fetal bovine serum (FBS), 1% L-glutamineand 1% antibiotic-antimycotic (100 units/mL of penicillin, 100 μg/mL ofstreptomycin, and 0.25 μg/mL of amphotericin B).

Cellular Uptake Studies For Protein Delivery and Endosomal Escape:

Cell internalization studies were performed with HeLa cells seeded at150,000 cells/mL in glass-bottomed petri-dishes and cultured for 24 h at37° C. in a 5% CO₂ incubator. Prior to delivery, cells were washed threetimes with PBS buffer and incubated with 1 mL media containingpolymer-rhodamine B-protein conjugate or rhodamine B-protein conjugate(protein concentration 30 μg/mL) at 37° C. for 6 h. After that, cellnucleus was stained with Hoechst 33342 (8 μM) and finally the media wasreplaced with fresh stock and incubated for another 1 h beforesubjecting to CLSM analysis. In addition, to study the endosomal escapeof the labelled proteins, HeLa cells were incubated with labellednanoassemblies for 4 and 24 h. After that it was stained withLysoTracker® Green to label endosomes/lysosomes and studied theco-localization of red and green fluorescence channels. Live cellimaging was performed using Nikon Spectral A1 confocal microscope.

Cell Viability With MTT Assay:

Different cells (including HeLa cells, MDA-MB-231, MCF-7) were seededinto 96-well tissue culture plates at a density of 10,000 cells/well/100μL sample and incubated at 37° C. After 24 h, culture media was replacedand cells were treated with different concentrations of protein-polymercomplex and control protein samples (0.1 mg/mL to 2 mg/mLprotein-polymer complex; for naked protein and modified proteinconcentration were matched with the protein-polymer complex) in 100 μLmedia (10 μL protein containing solution with differentconcentrations+90 μL medium). At the desired time interval, medium wasremoved and the cells were cultured by 100 μL 10% MTT (5 mg/mL) inmedium solution for another 4 h. Then, the solution was discarded andthe remaining crystal was dissolved by 100 μL DMSO. The solution wassubjected to absorbance measurement with SpectraMax® M5 at 590 nm. Celldeath was measured by the MTT assay in triplicate.

For ROS Sensitive PMA Treatment Enhanced Cytotoxicity

HeLa cells were seeded in a 96-well plate 24 h before the deliveryexperiment at a density of 10,000 cells per well (100 μL). On the day ofthe experiment, cells were pre-treated with RNase A-BA@polymernanoparticles with varied concentrations for 24 h. After nanoparticleremoval, cells were then treated with 200 nM PMA in DMEM or DMEM only asa control for 1 h. After removing PMA and washing the cells with DMEM,cells were maintained for another 24 h with fresh culture medium beforeviability measurement using the MTT assay. The toxicity of PMA wasexcluded by treating cells with 200 nM PMA only in the absence of RNaseA complex.

Materials, compositions, and components disclosed herein can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat when combinations, subsets, interactions, groups, etc. of thesematerials are disclosed that while specific reference of each variousindividual and collective combinations and permutations of thesecompounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including in the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps inmethods using the disclosed compounds or compositions. Thus, if thereare a variety of additional steps that can be performed, it isunderstood that each of these additional steps can be performed with anyspecific method steps or combination of method steps of the disclosedmethods, and that each such combination or subset of combinations isspecifically contemplated and should be considered disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. General principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 2006.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. When a range of values is listed, it isintended to encompass each value and sub-range within the range. Forexample, “C₁₋₆ alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆,C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆, C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄,C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —C(═O)—O— is equivalent to —O—C(═O)—.

Structures of compounds of the invention are limited by principles ofchemical bonding known to those skilled in the art. Accordingly, where agroup may be substituted by one or more of a number of substituents,such substitutions are selected so as to comply with principles ofchemical bonding and to give compounds that are not inherently unstableand/or would be known to one of ordinary skill in the art as likely tobe unstable under ambient conditions (e.g., aqueous, neutral, andseveral known physiological conditions).

Applicant's disclosure is described herein in preferred embodiments withreference to the figures, in which like numbers represent the same orsimilar elements. Reference throughout this specification to “oneembodiment,” “an embodiment,” or similar language means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” and similar language throughout this specificationmay, but do not necessarily, all refer to the same embodiment.

The described features, structures, or characteristics of Applicant'sdisclosure may be combined in any suitable manner in one or moreembodiments. In the description, herein, numerous specific details arerecited to provide a thorough understanding of embodiments of theinvention. One skilled in the relevant art will recognize, however, thatApplicant's composition and/or method may be practiced without one ormore of the specific details, or with other methods, components,materials, and so forth. In other instances, well-known structures,materials, or operations are not shown or described in detail to avoidobscuring aspects of the disclosure.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

Equivalents

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

1-20. (canceled)
 21. A polymer-protein conjugate, comprising: acopolymer comprising a first monomer of PEG-methacrylate (PEG-MA) and asecond monomer of methacrylate having a side chain conjugated to aprotein via a degradable linker.
 22. The polymer-protein conjugate ofclaim 21, wherein the degradable linker is sensitive to reactive oxygenspecies, a reducing environment, or change in pH.
 23. Thepolymer-protein conjugate of claim 22, wherein the degradable linkercomprises a boronate ester linker.
 24. The polymer-protein conjugate ofclaim 22, wherein the degradable linker comprises a disulfide linker.25. The polymer-protein conjugate of claim 22, wherein the degradablelinker comprises a cis-aconityl linker.
 26. A molecular assemblycomprising the polymer-protein conjugate of claim
 21. 27. The molecularassembly of claim 26, wherein the polymer:protein ratio by weight is inthe range from about 1:1 to about 50:1.
 28. The molecular assembly ofclaim 26, wherein the polymer-protein conjugate is adapted to releasethe protein for its native function upon degradation of the degradablelinker.
 29. The molecular assembly of claim 28, wherein thepolymer-protein conjugate is adapted to release the protein in thepresence of a specific and biologically relevant stimulus inside cells.30. The molecular assembly of claim 29, wherein the polymer-proteinconjugate is adapted to release the protein in the presence of aspecific and biologically relevant stimulus in the cytosol.
 31. Themolecular assembly of claim 27, wherein the protein has a molecularweight in the range of about 10 kDa to about 500 kDa.
 32. The molecularassembly of claim 31, wherein the protein has an isoelectric points (pI)in the range of about 3.0 to about 12.0.
 33. A composition comprisingthe molecular assembly of claim
 26. 34-54. (canceled)